Electrochemistry and electrogenerated chemiluminescence with a single faradaic electrode

ABSTRACT

Described herein is an apparatus comprising an electrochemical cell that employs a capacitive counter electrode and a faradaic working electrode. The capacitive counter electrode reduces the amount of redox products generated at the counter electrode while enabling the working electrode to generate redox products. The electrochemical cell is useful for controlling the redox products generated and/or the timing of the redox product generation. The electrochemical cell is useful in assay methods, including those using electrochemiluminescence. The electrochemical cell can be combined with additional hardware to form instrumentation for assay methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority as a divisional of U.S.patent application Ser. No. 13/485,662, filed on May 31, 2012, whichclaims the benefit of priority as a divisional of U.S. patentapplication Ser. No. 11/446,596, filed on Jun. 2, 2006, which claims thebenefit of priority to U.S. Provisional Patent Application No.60/686,935, filed on Jun. 3, 2005; and U.S. Provisional PatentApplication No. 60/695,163, filed on Jun. 30, 2005; and U.S. ProvisionalPatent Application No. 60/737,472, filed on Nov. 17, 2005, the completedisclosures of which are all incorporated herein by reference.

FIELD

This invention relates to an electrochemical apparatus comprising anelectrochemical cell with a faradaic working electrode and a capacitivecounter electrode and methods of the cell's use. The electrochemicalcell is useful for controlling the redox products generated and/or thetiming of the redox product generation. In some embodiments, it isuseful for generating electrochemiluminescence and methods of using thesame.

BACKGROUND

Electrochemiluminescent (ECL) methods and systems are useful in avariety of applications including medical diagnostics, food and beveragetesting, environmental monitoring, manufacturing quality control, drugdiscovery and basic scientific research. There are a number ofcommercially available instruments that utilize ECL for analyticalmeasurements.

Species that can be induced to emit ECL (ECL moieties) have been used asECL labels in various testing procedures. The light generated by ECLlabels can be used as a reporter signal in diagnostic procedures (Bardet al., U.S. Pat. No. 5,238,808). For instance, an ECL label can becovalently coupled to a binding agent such as an antibody or a nucleicacid probe, and the participation of the binding reagent in a bindinginteraction can be monitored by measuring ECL emitted from the ECLlabel. Alternatively, the ECL signal from an ECL-active compound can beindicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants).

For more background on ECL, ECL labels, ECL assays and instrumentationfor conducting ECL assays see U.S. Pat. Nos. 5,093,268; 5,147,806;5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519;5,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; 6,066,448;6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369; and 5,589,136 andPublished PCT Nos. WO99/63347; WO00/03233; WO99/58962; WO99/32662;WO99/14599; WO98/12539; WO97/36931 and WO98/57154.

Commercially available ECL instruments are widely used for reasonsincluding their excellent sensitivity, dynamic range, precision, andtolerance of complex sample matrices. Many commercially availableinstruments use flow cell-based designs with permanent reusable flowcells. The use of a permanent flow cell provides many advantages butalso some limitations, for example, in assay throughput. In someapplications, for example, the screening of chemical libraries forpotential therapeutic drugs, assay instrumentation should perform largenumbers of analyses at high speeds on small quantities of samples.

A variety of techniques have been developed for increasing assaythroughput and decreasing sample size. The use of multi-well assayplates allows for the parallel processing and analysis of multiplesamples distributed in multiple wells of a plate. Typically, samples andreagents are stored, processed and/or analyzed in multi-well assayplates (also known as microplates or microtiter plates). Multi-wellassay plates can take a variety of forms, sizes and shapes. Forconvenience, some standards have appeared for some instrumentation usedto process samples for high throughput assays. Multi-well assay platestypically are made in standard sizes and shapes and with standardarrangements of wells. Some well established arrangements of wellsinclude those found on 96-well plates (12×8 array of wells), 384-wellplates (24×16 array of wells) and 1536-well plate (48×32 array ofwells). The Society for Biomolecular Screening and ANSI have publishedmicroplate specifications for a variety of plate formats (see,http://www.sbsonline.org).

There is a need for ECL assay systems, and assays systems based on otherelectrochemical methods, that require lower sample volume, and are lessexpensive, faster, and more sensitive. As these assays move to thenanoscale to address these needs, it is increasingly difficult toseparate the working electrode from the counter electrode: As theworking and counter electrodes are brought closer together in the samecell, undesirable redox byproducts formed at the counter electrode caninteract with species at the working electrode.

To date, cells using one capacitive and one faradaic electrode have beenused in solid state systems, for example, to inject charge into thinlayers of luminescent organic polymers to aid the observation ofspectroscopic properties. In such systems, one electrode contacts thepolymer layer and the other electrode is a small tip separated from thepolymer layer by a ˜10 nm insulating layer of impurities or air. See forexample, Adams, et al., J. Phys. Chem. B, 2000, 104, 6728. These cellsare not electrochemical cells, do not use an electrolyte solution, andare not designed to contain an electrolyte solution. Configurationsinvolving a capacitive electrode and a reference electrode have beenused to examine double layer effects. See, for example, Grahame, Chem.Rev., 1947, 41, 441. These studies focus on the importance of thepolarized electrode. Any faradaic effects at the reference electrodewere not of interest and were neglected.

There remains a need for an electrochemical apparatus that reduces theintroduction of undesirable electrochemically generated byproducts intothe sample.

There remains a need for an electrochemical apparatus that separatelycontrols the timing of the generation of oxidation and reductionproducts.

SUMMARY

The present invention provides an apparatus comprising anelectrochemical cell comprising: a faradaic working electrode and acapacitive counter electrode wherein the electrochemical cell is capableof receiving an electrolyte solution that can simultaneously contactsaid faradaic working electrode and said capacitive counter electrode.

Also provided are methods of determining the presence or amount of ananalyte in a sample comprising the steps of:

(a) optionally preprocessing the sample;

(b) contacting a faradaic working electrode to a solution comprising theoptionally pre-processed sample; and an electrolyte;

(c) contacting a capacitive counter electrode to the solution;

(d) supplying electrical energy between the faradaic working electrodeand the capacitive counter electrode sufficient to provide for faradaiccharge transfer at the faradaic working electrode;

(e) measuring at least one of (i) light, (ii) current, (iii) voltage,and (iv) charge to determine the presence or amount of the analyte inthe sample.

Also provided are methods of generating at least one electrochemicalproduct at a working electrode while generating a discordantly smalleramount of electrochemical byproduct at a counter electrode, comprisingthe steps of:

contacting a faradaic working electrode with an electrolyte solution;

contacting a capacitive counter electrode with the electrolyte solution;and

applying electrical energy between the faradaic working electrode andthe capacitive counter electrode wherein the faradaic charge transferredacross the faradaic working electrode is at least-about 10 times thefaradaic charge transferred across the capacitive counter electrode,thus generating a discordantly smaller amount of electrochemicalbyproduct at the counter electrode.

In this method and any methods disclosed or claimed herein, the faradaicand the capacitive electrodes may be placed in contacted with theelectrolyte or sample solution in any order.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram showing the system Pt/water/SiO₂/Si with thepossibility of lateral tip movement to generate fresh contact surface.The tip was attached to a translation stage that could be moved with aninchworm motor, a micrometer, or by hand.

FIG. 2. Charging current as a function of time in a system ofPt/water/SiO₂/Si under a bias of 1 V applied between Pt and Si. Afterthe charging current reached baseline, the circuit was disconnected forabout 8 s and reconnected again still under the same bias; only a verysmall charging current was seen as shown in the inset.

FIG. 3. Charging current recorded during the relative movement under abias of −1 V applied between Pt and Si in a system of Pt/water/SiO₂/Si.Lateral scan rate, ˜1 cm/s. Inset: Charging current as a function ofbias with stationary tip at a scan rate of 100 mV/s.

FIG. 4. Charging current, as in FIG. 3 during repeated steps, i.e., astop-and-go lateral movement, under a constant bias of −1 V between Ptand Si. The current increased as the water drop movement started anddecreased when the movement stopped, with a brief steady state chargingcurrent during the movement.

FIG. 5. Charging current (bottom) and electrogenerated chemiluminescence(top) produced in the system of Pt/water/SiO₂/Si with continuous appliedpotential pulses from 1.4 V to −0.5 V between Pt and Si, in aqueous 0.5mM Ru(bpy)₃ ²⁺, 0.10 M tripropylamine (TPA) and 0.10 M Tris/0.10 MLiClO₄ buffer (pH=8). Inset: Micrograph of an ECL image from a 25 μm Pttip obtained with an optical microscope in a separate experiment withthe same system under a bias of 1.4 V.

FIG. 6. Schematic diagram showing a motor driven, moving, blockedcounter electrode, e.g. a conductive wire or tape coated with aninsulating layer, through a solution containing an electrolyte under abias to produce a continuous faradaic reaction at the working electrode.

DETAILED DESCRIPTION

The following description refers to the accompanying drawings in which,in the absence of a contrary representation, the same numbers indifferent drawings represent similar elements. The implementations inthe following description do not represent all implementationsconsistent with principles of the claimed invention. Instead, they aremerely some examples of systems and methods consistent with thoseprinciples. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

I. Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise. These definitions are placed in this section for the reader'sconvenience. Definitions for other terms, words and phrases, which maybe found in other sections of this application, also describe theintended meanings, except to the extent that the context in which theyare used indicates otherwise.

A. General Definitions

The term “aliphatic,” as used herein, is defined as in The AmericanHeritage® Dictionary of the English Language, Fourth Edition Copyright ©2000 and encompasses organic chemical compounds in which the carbonatoms are linked in open chains. The open chains comprise from 1 to 20carbon atoms, or from 1 to 13, or from 1 to 6 carbon atoms. When analiphatic group is unsaturated there can be from 1 to 10, or from 1 to6, or from 1 to 3 points of unsaturation. The number of carbon atoms inan aliphatic group can be indicated by a subscript on a “C” (forexample, “C₃ aliphatic” represents an aliphatic group comprising 3carbon atoms). Likewise, ranges can be expressed in the subscript. Forexample “C₁₋₁₀ aliphatic” encompasses aliphatic groups of from 1 to 10carbon atoms inclusive. Examples of aliphatic groups include, but arenot limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl,tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl,3-hexyl, 3-methylpentyl, ethene, propene, ethyne, butene, propyne,butyne, and the like. When an aliphatic group having a specific numberof carbons is named using the subscripted C notation, all isomers havingthat number of carbons are intended to be encompassed. Aliphatic groupscan be optionally substituted by at least one hydrophilic functionalgroup, as defined herein. In addition, aliphatic groups useful as ECLmoieties and as ECL coreactants may also comprise additional functionalgroups and may have a single (i.e., monodentate ligand) or multiple(i.e., bidentate or polydentate ligands) points of attachment. Suchaliphatic groups are well known in the art and are described inElectrogenerated Chemiluminescence, Bard, Editor, Marcel. Dekker,(2004); Knight, A and Greenway, G. Analyst 119:879-890 1994.

The term “hydrophilic functional group” refers to a functional groupthat facilitates or that increases the solubility of a molecule inwater. Examples include, but are not limited to, groups such as hydroxyl(—OH), aldehyde (—C(O)H), hydroxycarbonyl (—C(OH)(C═O)H), amino (—NH₂),aminocarbonyl (—CONH₂), amidine (—C(═NH)NH₂), imino (—C═NH), cyano(—CN), nitro (—NO₂), nitrate (—NO₃), sulfate (—SO₄), sulfonate (—SO₃H),phosphate (—PO₄), phosphonate (—PH₂O₃), silicate (—SiHO₃), carboxylate(—COOH), borate (—BH₂O₃), guanidinium (—HN—C(═NH)—NH₂), carbamide(—HNC(O)NH₂), carbamate (—HNC(O)NH₂), carbonate (—CO₃), sulfamide(—S(O)₂NH₂), silyl (—SiH₃ and/or —Si(OH)₃), siloxy (—OSiH₃ and/or—OSi(OH)₃), amide and the like.

The term “binding partner,” as used herein, means a substance that canbind specifically to an analyte of interest. In general, specificbinding is characterized by a relatively high affinity and a relativelylow to moderate capacity. Nonspecific binding usually has a low affinitywith a moderate to high capacity. Typically, binding is consideredspecific when the affinity constant Ka is higher than about 10⁶ M⁻¹. Forexample, binding may be considered specific when the affinity constantKa is higher than about 10⁸M⁻¹. A higher affinity constant indicatesgreater affinity, and thus typically greater specificity. For example,antibodies typically bind antigens with an affinity constant in therange of 10⁶M⁻¹ to 10⁹M⁻¹ or higher.

Examples of binding partners include complementary nucleic acidsequences (e.g., two DNA sequences which hybridize to each other; twoRNA sequences which hybridize to each other; a DNA and an RNA sequencewhich hybridize to each other), an antibody and an antigen, a receptorand a ligand (e.g., TNF and TNFr-I, CD142 and Factor VIIa, B7-2 andCD28, HIV-1 and CD4, ATR/TEM8 or CMG and the protective antigen moietyof anthrax toxin), an enzyme and a substrate, or a molecule and abinding protein (e.g., vitamin B12 and intrinsic factor, folate andfolate binding protein).

As mentioned above, antibodies are an example of a binding partner. Theterm “antibody,” as used herein, means an immunoglobulin or a partthereof, and encompasses any polypeptide (with or without furthermodification by sugar moieties (mono and polysaccharides)) comprising anantigen binding site regardless of the source, method of production, orother characteristics. The term includes, for example, polyclonal,monoclonal, monospecific, polyspecific, humanized, single chain,chimeric, synthetic, recombinant, hybrid, mutated, and CDR graftedantibodies as well as fusion proteins. A part of an antibody can includeany fragment which can bind antigen, including but not limited to Fab,Fab′, F(ab′)₂, Facb, Fv, ScFv, Fd, V_(H), and V_(L).

A large number of monoclonal antibodies that bind to various analytes ofinterest are available, as exemplified by the listings in variouscatalogs, such as: Biochemicals and Reagents for Life Science Research,Sigma-Aldrich Co., P.O. Box 14508, St. Louis, Mo., 63178 (1999); theLife Technologies Catalog, Life Technologies, Gaithersburg, Md.; and thePierce Catalog, Pierce Chemical Company, P.O. Box 117, Rockford, Ill.61105 (1994).

Other exemplary monoclonal antibodies include those that bindspecifically to β-actin, DNA, digoxin, insulin, progesterone, humanleukocyte markers, human interleukin-10, human interferon, humanfibrinogen, p53, hepatitis B virus or a portion thereof, HIV virus or aportion thereof, tumor necrosis factor, or FK-506. In certainembodiments, the monoclonal antibody is chosen from antibodies that bindspecifically to at least one of T4, T3, free T3, free T4, TSH(thyroid-stimulating hormone), thyroglobulin, TSH receptor, prolactin,LH (luteinizing hormone), FSH (follicle stimulating hormone),testosterone, progesterone, estradiol, hCG (human ChorionicGondaotropin), hCG+β, SHBG (sex hormone-binding globulin), DHEA-S(dehydroepiandrosterone sulfate), hGH (human growth hormone), ACTH(adrenocorticotropic hormone), cortisol, insulin, ferritin, folate, RBC(red blood cell) folate, vitamin B12, vitamin D, C-peptide, troponin T,CK MB (creatine kinase-myoglobin), myoglobin, pro-BNP (brain natriureticpeptide), HbsAg (hepatitis B surface antigen), HbeAg (hepatitis Beantigen), HIV antigen, HIV combined, H. pylori, β-CrossLaps,osteocalcin, PTH (parathyroid hormone), IgE, digoxin, digitoxin, AFP(α-fetoprotein), CEA (carcinoembryonic antigen), PSA (prostate specificantigen), free PSA, CA (cancer antigen) 19-9, CA 12-5, CA 72-4, cyfra21-1, NSE (neuron specific enolase), S-100, P1NP (procollagen type 1N-propeptide), PAPP-A (pregnancy-associated plasma protein-A), Lp-PLA2(lipoprotein-associated phospholipase A2), sCD40L (soluble CD40 Ligand),IL 18, and Survivin.

Other exemplary monoclonal antibodies include anti-TPO (antithyroidperoxidase antibody), anti-HBc (Hepatitis Bc antigen), anti-HBc/IgM,anti-HAV (hepatitis A virus), anti-HAV/IgM, anti-HCV (hepatitis Cvirus), anti-HIV, anti-HIV p-24, anti-rubella IgG, anti-rubella IgM,anti-toxoplasmosis IgG, anti-toxoplasmosis IgM, anti-CMV(cytomegalovirus) IgG, anti-CMV IgM, anti-HGV (hepatitis G virus), andanti-HTLV (human T-lymphotropic virus).

Further examples of binding partners include binding proteins, forexample, vitamin B12 binding protein, DNA binding proteins such as thesuperclasses of basic domains, zinc-coordinating DNA binding domains,Helix-turn-helix, beta scaffold factors with minor groove contacts, andother transcription factors that are not antibodies.

The term “labeled binding partner,” as used herein, means a bindingpartner that is labeled with an atom, moiety, functional group, ormolecule capable of generating, modifying or modulating a detectablesignal. For example, in a radiochemical assay, the labeled bindingpartner may be labeled with a radioactive isotope of iodine.Alternatively, the labeled binding partner antibody may be labeled withan enzyme—e.g., horseradish peroxidase—that can be used in acolorimetric assay. The labeled binding partner may also be labeled witha fluorophore, such as one useful in fluorescence measurements,time-resolved fluorescence measurements or a fluorescence resonanceenergy transfer (FRET) measurements. Exemplary reporters are disclosedin Hemmila, et al., J. Biochem. Biophys. Methods, vol. 26, pp. 283-290(1993); Kakabakos, et al., Clin. Chem., vol. 38, pp. 338-342 (1992); Xu,et al., Clin. Chem., pp. 2038-2043 (1992); Hemmila, et al., Scand. J.Clin. Lab. Invest., vol. 48, pp. 389-400 (1988); Bioluminescence andChemiluminescence Proceedings of the 9th International Symposium 1996,J. W. Hastings, et al., Eds., Wiley, New York, 1996; Bioluminescence andChemiluminescence Instruments and Applications, Knox Van Dyre, Ed., CRCPress, Boca Raton, 1985; I. Hemmila, Applications of Fluorescence inImmunoassays, Chemical Analysis, Volume 117, Wiley, New York, 1991; andBlackburn, et al., Clin. Chem., vol. 37, p. 1534 (1991).

Further examples of labeled binding partners include binding partnersthat are labeled with an electrochemiluminescent moiety (ECL moiety),functional group, or molecule that is useful for generating a signal inan electrochemiluminescent (ECL) assay. The ECL moiety may be anycompound that can be induced to repeatedly emit electromagneticradiation by direct exposure to an electrical energy source. Suchmoieties, functional groups, or molecules are disclosed in U.S. Pat.Nos. 5,962,218; 5,945,344; 5,935,779; 5,858,676; 5,846,485; 5,811,236;5,804,400; 5,798,083; 5,779,976; 5,770,459; 5,746,974; 5,744,367;5,731,147; 5,720,922; 5,716,781; 5,714,089; 5,705,402; 5,700,427;5,686,244; 5,679,519; 5,643,713; 5,641,623; 5,632,956; 5,624,637;5,610,075; 5,597,910; 5,591,581; 5,543,112; 5,466,416; 5,453,356;5,310,687; 5,296,191; 5,247,243; 5,238,808; 5,221,605; 5,189,549;5,147,806; 5,093,268; 5,068,088; 5,061,445; and 6,808,939; Dong, L. etal., Anal. Biochem., vol. 236, pp. 344-347 (1996); Blohm, et al.,Biomedical Products, vol. 21, No. 4:60 (1996); Jameison, et al., Anal.Chem., vol. 68, pp. 1298-1302 (1996); Kibbey, et al., NatureBiotechnology, vol. 14, no. 3, pp. 259-260 (1996); Yu, et al., Appliedand Environmental Microbiology, vol. 62, no. 2, pp. 587-592 (1996);Williams, American Biotechnology, p. 26—(January, 1996); Darsley, etal., Biomedical Products, vol. 21, no. 1, p. 133 (January, 1996);Kobrynski, et al., Clinical and Diagnostic Laboratory Immunology, vol.3, no. 1, pp. 42-46 (January 1996); Williams, IVD Technology, pp. 28-31(November, 1995); Deaver, Nature, vol. 377, pp. 758-760 (Oct. 26, 1995);Yu, et al., BioMedical Products, vol. 20, no. 10, p. 20 (October, 1995);Kibbey, et al., BioMedical Products, vol. 20, no. 9, p. 116 (September,1995); Schutzbank, et al., Journal of Clinical Microbiology, vol. 33,pp. 2036-2041 (August, 1995); Stern, et al., Clinical Biochemistry, vol.28, pp. 470-472 (August, 1995); Carlowicz, Clinical Laboratory News,vol. 21, no. 8, pp. 1-2 (August 1995); Gatto-Menking, et al., Biosensors& Bioelectronics, vol. 10, pp. 501-507 (July, 1995); Yu, et al., Journalof Bioluminescence and Chemiluminescence, vol. 10, pp. 239-245 (1995);Van Gemen, et al., Journal of Virology Methods, vol. 49, pp. 157-168(1994); Yang, et al., Bio/Technology, vol. 12, pp. 193-194 (1994);Kenten, et al., Clinical Chemistry, vol. 38, pp. 873-879 (1992); Kenten,Non-radioactive Labeling and Detection of Biomolecules, Kessler, Ed.,Springer, Berlin, pp. 175-179 (1992); Gudibande, et al., Journal ofMolecular and Cellular Probes, vol. 6, pp. 495-503 (1992); Kenten, etal., Clinical Chemistry, vol. 37, pp. 1626-1632 (1991); Blackburn, etal., Clinical Chemistry, vol. 37, pp. 1534-1539 (1991), ElectrogeneratedChemiluminescence, Bard, Editor, Marcel Dekker, (2004), and U.S. Pat.No. 5,935,779. In certain embodiments, the electrochemiluminescent groupcan comprise a metal, such as ruthenium or osmium. In certainembodiments, the binding partner can be labeled with a ruthenium moiety,such as a tris-bipyridyl-ruthenium group such as ruthenium (II)tris-bipyridine ([Ru(bpy)₃]²⁺).

The term “analyte,” as used herein, means any molecule, or aggregate ofmolecules, including a cell or a cellular component of a virus, found ina sample. Examples of analytes to which the binding partner canspecifically bind include bacterial toxins, viruses, bacteria, proteins,hormones, DNA, RNA, drugs, antibiotics, nerve toxins, and metabolitesthereof. Also included in the scope of the term “analyte” are fragmentsof any molecule found in a sample. An analyte may be an organiccompound, an organometallic compound or an inorganic compound. Ananalyte may be a nucleic acid (e.g., DNA, RNA, a plasmid, a vector, oran oligonucleotide), a protein (e.g., an antibody, an antigen, areceptor, a receptor ligand, or a peptide), a lipoprotein, aglycoprotein, a ribo- or deoxyribonucleoprotein, a peptide, apolysaccharide, a lipopolysaccharide, a lipid, a fatty acid, a vitamin,an amino acid, a pharmaceutical compound (e.g., tranquilizers,barbiturates, opiates, alcohols, tricyclic antidepressants,benzodiazepines, anti-virals, anti-fungals, antibiotics, steroids,cardiac glycosides, or a metabolite of any of the preceding), a hormone,a growth factor, an enzyme, a coenzyme, an apoenzyme, a hapten, alectin, a substrate, a cellular metabolite, a cellular component ororganelle (e.g., a membrane, a cell wall, a ribosome, a chromosome, amitochondria, or a cytoskeleton component). Also included in thedefinition are toxins, pesticide, herbicides, and environmentalpollutants. The definition further includes complexes comprising one ormore of any of the examples set forth within this definition.

Further examples of analytes include bacterial pathogens such asAeromonas hydrophile and other species (spp.); Bacillus anthracis;Bacillus cereus; Botulinum neurotoxin producing species of Clostridium;Brucella abortus; Brucella melitensis; Brucella suis; Burkholderiamallei (formally Pseudomonas mallei); Burkholderia pseudomallei(formerly Pseudomonas pseudomallei); Campylobacter jejuni; Chlamydiapsittaci; Clostridium botulinum; Clostridium perfringens; Cowdriaruminantium (Heartwater); Coxiella burnetii; Enterovirulent Escherichiacoli group (EEC Group) such as Escherichia coli—enterotoxigenic (ETEC),Escherichia coli—enteropathogenic (EPEC), Escherichia coli—O157:H7enterohemorrhagic (EHEC), and Escherichia coli—enteroinvasive (EIEC);Ehrlichia spp. such as Ehrlichia chaffeensis; Francisella tularensis;Legionella pneumophilia; Liberobacter africanus; Liberobacter asiaticus;Listeria monocytogenes; miscellaneous enterics such as Klebsiella,Enterobacter, Proteus, Citrobacter, Aerobacter, Providencia, andSerratia; Mycobacterium bovis; Mycobacterium tuberculosis; Mycoplasmacapricolum; Mycoplasma mycoides ssp mycoides; Peronosclerosporaphilippinensis; Phakopsora pachyrhizi; Plesiomonas shigelloides;Ralstonia solanacearum race 3, biovar 2; Rickettsia prowazekii;Rickettsia rickettsii; Salmonella spp.; Schlerophthora rayssiae varzeae; Shigella spp.; Staphylococcus aureus; Streptococcus; Synchytriumendobioticum; Vibrio cholerae non-O1; Vibrio cholerae O1; Vibrioparahaemolyticus and other Vibrios; Vibrio vulnificus; Xanthomonasoryzae; Xylella fastidiosa (citrus variegated chlorosis strain);Yersinia enterocolitica and Yersinia pseudotuberculosis; and Yersiniapestis.

Further examples of analytes include viruses such as African horsesickness virus; African swine fever virus; Akabane virus; Avianinfluenza virus (highly pathogenic); Bhanja virus; Blue tongue virus(Exotic); Camel pox virus; Cercopithecine herpesvirus 1; Chikungunyavirus; Classical swine fever virus; Coronavirus (SARS); Crimean-Congohemorrhagic fever virus; Dengue viruses; Dugbe virus; Ebola viruses;Encephalitic viruses such as Eastern equine encephalitis virus, Japaneseencephalitis virus, Murray Valley encephalitis, and Venezuelan equineencephalitis virus; Equine morbillivirus; Flexal virus; Foot and mouthdisease virus; Germiston virus; Goat pox virus; Hantaan or other Hantaviruses; Hendra virus; Issyk-kul virus; Koutango virus; Lassa fevervirus; Louping-ill virus; Lumpy skin disease virus; Lymphocyticchoriomeningitis virus; Malignant catarrhal fever virus (Exotic);Marburg virus; Mayaro virus; Menangle virus; Monkeypox virus; Mucambovirus; Newcastle disease virus (VVND); Nipah Virus; Norwalk virus group;Oropouche virus; Orungo virus; Peste Des Petits Ruminants virus; Piryvirus; Plum Pox Potyvirus; Poliovirus; Potato virus; Powassan virus;Rift Valley fever virus; Rinderpest virus; Rotavirus; Semliki Forestvirus; Sheep pox virus; South American hemorrhagic fever viruses such asFlexal, Guanarito, Junin, Machupo, and Sabia; Spondweni virus; Swinevesicular disease virus; Tick-borne encephalitis complex (flavi) virusessuch as Central European tick-borne encephalitis, Far Eastern tick-borneencephalitis, Russian spring and summer encephalitis, Kyasanur forestdisease, and Omsk hemorrhagic fever; Variola major virus (Smallpoxvirus); Variola minor virus (Alastrim); Vesicular stomatitis virus(Exotic); Wesselbron virus; West Nile virus; Yellow fever virus; virusesfrom the family papovaviridae, including polyomaviruses such as SV40, JCand BK and including papillomaviruses (e.g., HPV); parvoviruses (e.g.,B19 and RA-1); and viruses from the family Picornaviridae, includingrhinoviruses and Coxsackie B. Other viruses that can comprise a targetnucleic acid sequence include species not mentioned above belonging tothe families Adenoviridae, Arenaviridae, Arterivirus, Astroviridae,Baculoviridae, Badnavirus, Bamaviridae, Birnaviridae, Bromoviridae,Bunyaviridae, Caliciviridae, Capillovirus, Carla virus, Caulimovirus,Circoviridae, Closterovirus, Comoviridae, Coronaviridae, Corticoviridae,Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae,Flaviviridae, Furovirus, Fuselloviridae, Geminiviridae, Hepadnaviridae,Herpesviridae, Hordeivirus, Hypoviridae, Idaeovirus, Inoviridae,Iridoviridae, Leviviridae, Lipothrixviridae, Luteovirus, Machlomovirus,Marafivirus, Microviridae, Myoviridae, Necrovirus, Nodaviridae,Orthomyxoviridae, Paramyxoviridae, Partitiviridae, Parvoviridae,Phycodnaviridae, Plasmaviridae, Podoviridae, Polydnaviridae, Potexvirus,Potyviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae,Rhizidiovirus, Sequiviridae, Siphoviridae, Sobemovirus, Tectiviridae,Tenuivirus, Tetraviridae, Tobamovirus, Tobravirus, Togaviridae,Tombusviridae, Totiviridae, Tymovirus, and Umbra virus.

Further examples of analytes include toxins such as Abrin; Aflatoxins;Botulinum neurotoxin; Ciguatera toxins; Clostridium perfringens epsilontoxin; Conotoxins; Diacetoxyscirpenol; Diphtheria toxin; Grayanotoxin;mushroom toxins such as amanitins, gyromitrin, and orellanine;Phytohaemagglutinin; Pyrrolizidine alkaloids; Ricin; shellfish toxins(paralytic, diarrheic, neurotoxic or amnesic) such as saxitoxin, akadaicacid, dinophysis toxins, pectenotoxins, yessotoxins, brevetoxins, anddomoic acid; Shigatoxins; Shiga-like ribosome inactivating proteins;snake toxins; Staphylococcal enterotoxins; T-2 toxin; and Tetrodotoxin.

Further examples of analytes include prion proteins such as the Bovinespongiform encephalopathy agent.

Further examples of analytes include parasitic protozoa and worms, suchas: Acanthamoeba and other free-living amoebae; Anisakis sp. and otherrelated worms Ascaris lumbricoides and Trichuris trichiura;Cryptosporidium parvum; Cyclospora cayetanensis; Diphyllobothrium spp.;Entamoeba histolytica; Eustrongylides spp.; Giardia lamblia; Nanophyetusspp.; Shistosoma spp.; Toxoplasma gondii; and Trichinella.

Further examples of analytes include fungi such as: Aspergillus spp.;Blastomyces dermatitidis; Candida; Coccidioides immitis; Coccidioidesposadasii; Cryptococcus neoformans; Histoplasma capsulatum; Maize rust;Rice blast; Rice brown spot disease; Rye blast; Sporothrix schenckii;and wheat fungus.

Further examples of analytes include genetic elements, recombinantnucleic acids, and recombinant organisms, such as

(1) nucleic acids (synthetic or naturally derived, contiguous orfragmented, in host chromosomes or in expression vectors) that canencode infectious and/or replication competent forms of any of theselect agents.

(2) nucleic acids (synthetic or naturally derived) that encode thefunctional form(s) of any of the toxins listed if the nucleic acids

-   -   (i) are in a vector or host chromosome;    -   (ii) can be expressed in vivo or in vitro; or    -   (iii) are in a vector or host chromosome and can be expressed in        vivo or in vitro;

(3) nucleic acid—protein complexes that are locations of cellularregulatory events:

-   -   (i) viral nucleic acid—protein complexes that are precursors to        viral replication;    -   (ii) RNA-protein complexes that modify RNA structure and        regulate protein transcription events; or    -   (iii) Nucleic acid—protein complexes that are regulated by        hormones or secondary cell signaling molecules.

(4) viruses, bacteria, fungi, and toxins that have been geneticallymodified.

Further examples of analytes include immune response molecules to theabove-mentioned analyte examples such as IgA, IgD, IgE, IgG, and IgM.

The term “analog of the analyte,” as used herein, refers to a substancethat competes with the analyte of interest for binding to a bindingpartner. An analog of the analyte may be a known amount of the analyteof interest itself that is added to compete for binding to a specificbinding partner with analyte of interest present in a sample. Examplesof analogs of the analyte include azidothymidine (AZT), an analog of anucleotide which binds to HIV reverse transcriptase, puromycin, ananalog of the terminal aminoacyl-adenosine part of aminoacyl-tRNA, andmethotrexate, an analog of tetrahydrofolate. Other analogs may bederivatives of the analyte of interest. The term “labeled analog of theanalyte”, as used herein, is defined in an analogous manner to labeledbinding partner.

As used herein, the term “support,” refers to any of the ways forimmobilizing binding partners that are known in the art, such asmembranes, beads, particles, electrodes, or even the walls or-surfacesof a container. The support may comprise any material on which thebinding partner is conventionally immobilized, such as nitrocellulose,polystyrene, polypropylene, polyvinyl chloride, EVA, glass, carbon,glassy carbon, carbon black, carbon nanotubes or fibrils, platinum,palladium, gold, silver, silver chloride, iridium, rhodium, or alloyscomprising the metallic elements in this list. In one embodiment, thesupport is a bead, such as a polystyrene bead or a magnetizable bead. Asused herein, the term “magnetizable bead” encompasses magnetic,paramagnetic, and superparamagnetic beads and/or particles. In variousembodiments, beads can have a number of different sizes, for example,larger than about 3 mm, smaller than about 3 mm, smaller than about 1mm, smaller than about 0.1 mm, smaller than about 100 μm, smaller thanabout 10 μm, smaller than about 5 μm, about 2.8 μm, about 1 μm, smallerthan about 1 μm, smaller than about 0.5 μm, about 0.1 μm, or smallerthan about 0.1 μm. Bead size can range from about 0.1 μm to about 5 mm,or from about 0.1 μm to about 3 mm, or from about 1 μm to about 3 μm, orfrom about 2.8 μm to about 5 μm. Combinations of beads sizes can also beused. In one embodiment, the support is a microcentrifuge tube or atleast one well of a multiwell plate.

The term “dry composition,” as used herein, means that the compositionhas a moisture content of less than or equal to about 5% by weight,relative to the total weight of the composition. Examples of drycompositions include compositions that have a moisture content of lessthan or equal to about 3% by weight relative to the total weight of thecomposition, of less than or equal to about 1% by weight relative to thetotal weight of the composition and compositions that have a moisturecontent ranging from about 1% to about 3% by weight, relative to thetotal weight of the composition.

In some embodiments of the present invention, theassay-performance-substance is a dry composition. In some embodiments ofthe present invention, the ECL moiety is a dry composition.

The term “sample,” as used herein, comprises liquids that can containthe analyte. The term “liquid,” as used herein comprises-in addition tothe more traditional definition of liquid-colloids, suspensions,slurries, and dispersions of particles in a liquid wherein the particleshave a sedimentation rate due to earth's gravity of less than about 1mm/s. The sample can be drawn from any source upon which analysis isdesired. For example, the sample can arise from body or other biologicalfluid, such as blood, plasma, serum, milk, semen, amniotic fluid,cerebral spinal fluid, sputum, bronchoalveolar lavage, tears, saliva,urine, or stool. Alternatively, the sample can be a water sampleobtained from a body of water, such as lake or river. The sample canalso be prepared by dissolving or suspending a sample in a liquid, suchas water or an aqueous buffer. The sample source can be a surface swab;for example, a surface can be swabbed; the swab washed by a liquid;thereby transferring an analyte from the surface into the liquid. Thesample source can be air; for example, the air can be filtered; thefilter washed by a liquid; thereby transferring an analyte from the airinto the liquid.

The term “sample matrix,” as used herein, refers to everything in thesample with the exception of the analyte.

The term “magnetic field source,” as used herein, includes permanentmagnets and electromagnets, which are separate, individual entities withdefined N-S magnetic poles. A “dipole magnet” comprises one magneticfield source.

The term “sandwich magnet,” as used herein, refers to magnets comprisingtwo or more magnetic field sources configured such that their opposingmagnetic fields overlap or are coerced. This can be accomplished byplacing opposing poles (N-N or S-S) in closer proximity to each otherthan the attracting poles (N-S) of the magnetic fields sources. Forexample, two dipole magnets arranged in a N-S-S-N or a S-N-N-Sconfiguration would form a sandwich magnet.

The term “channel magnet,” as used herein, refers to a single magneticfield source bonded to a highly magnetizable material in the form of aU-shaped channel. In such a configuration, the magnetizable materialbecomes an extension of the magnetic pole to which it is bound.

B. ECL Moieties

The term “ECL moiety” refers to an electrochemiluminescent moiety, whichis any compound that can be induced to repeatedly emit electromagneticradiation by exposure to an electrical energy source. Representative ECLmoieties are described in Electrogenerated Chemiluminescence, Bard,Editor, Marcel Dekker, (2004); Knight, A and Greenway, G. Analyst119:879-890 1994; and in U.S. Pat. Nos. 5,221,605; 5,591,581; 5,858,676;and 6,808,939. Preparation of primers comprising ECL moieties is wellknown in the art, as described, for example, in U.S. Pat. No. 6,174,709.Some ECL moieties emit electromagnetic radiation is the visible spectrumwhile other might emit other types of electromagnetic radiation, such asinfrared or ultraviolet light, X-rays, microwaves, etc. Use of the terms“electrochemiluminescence”, “electrochemiluminescent”,“electrochemiluminesce”, “luminescence”, “luminescent” and “luminesce”in connection with the present invention does not require that theemission be light, but includes the emission being such other forms ofelectromagnetic radiation.

ECL moieties can be transition metals. For example, the ECL moiety cancomprise a metal-containing organic compound wherein the metal can bechosen, for example, from ruthenium, osmium, rhenium, iridium, rhodium,platinum, palladium, molybdenum, and technetium. For example, the metalcan be ruthenium or osmium. For example, the ECL moiety can be aruthenium chelate or an osmium chelate. For example, the ECL moiety cancomprise bis(2,2′-bipyridyl)ruthenium(II) andtris(2,2′-bipyridyl)ruthenium(II). For example, the ECL moiety can beruthenium (II) tris bipyridine ([Ru(bpy)₃]²⁺). The metal can also bechosen, for example, from rare earth metals, including but not limitedto cerium, dysprosium, erbium, europium, gadolinium, holmium, lanthanum,lutetium, neodymium, praseodymium, promethium, terbium, thulium, andytterbium. For example, the metal can be cerium, europium, terbium, orytterbium.

Metal-containing ECL moieties can have the formula

M(P)_(m)(L1)_(n)(L2)_(o)(L3)_(p)(L4)_(q)(L5)_(r)(L6)_(s)

wherein M is a metal; P is a polydentate ligand of M; L1, L2, L3, L4, L5and L6 are ligands of M, each of which can be the same as, or differentfrom, each other; m is an integer equal to or greater than 1; each of n,o, p, q, r and s is an integer equal to or greater than zero; and P, L1,L2, L3, L4, L5 and L6 are of such composition and number that the ECLmoiety can be induced to emit electromagnetic radiation and the totalnumber of bonds to M provided by the ligands of M equals thecoordination number of M. For example, M can be ruthenium.Alternatively, M can be osmium.

Some examples of the ECL moiety can have one polydentate ligand of M.The ECL moiety can also have more than one polydentate ligand. Inexamples comprising more than one polydentate ligand of M, thepolydentate ligands can be the same or different. Polydentate ligandscan be aromatic or aliphatic ligands. Suitable aromatic polydentateligands can be aromatic heterocyclic ligands and can benitrogen-containing, such as, for example, bipyridyl, bipyrazyl,terpyridyl, 1,10 phenanthroline, and porphyrins.

Suitable polydentate ligands can be unsubstituted, or substituted by anyof a large number of substituents known to the art. Suitablesubstituents include, but are not limited to, alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate,carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, maleimidesulfur-containing groups, phosphorus-containing groups, and thecarboxylate ester of N-hydroxysuccinimide.

In some embodiments, at least one of L1, L2, L3, L4, L5 and L6 can be apolydentate aromatic heterocyclic ligand. In various embodiments, atleast one of these polydentate aromatic heterocyclic ligands can containnitrogen. Suitable polydentate ligands can be, but are not limited to,bipyridyl, bipyrazyl, terpyridyl, 1,10 phenanthroline, a porphyrin,substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl,substituted 1,10 phenanthroline or a substituted porphyrin. Thesesubstituted polydentate ligands can be substituted with an alkyl,substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, maleimidea sulfur-containing group, a phosphorus-containing group or thecarboxylate ester of N-hydroxysuccinimide.

Some ECL moieties can contain two bidentate ligands, each of which canbe bipyridyl, bipyrazyl, terpyridyl, 1,10 phenanthroline, substitutedbipyridyl, substituted bipyrazyl, substituted terpyridyl or substituted1,10 phenanthroline.

Some ECL moieties can contain three bidentate ligands, each of which canbe bipyridyl, bipyrazyl, terpyridyl, 1,10-phenanthroline, substitutedbipyridyl, substituted bipyrazyl, substituted terpyridyl or substituted1,10-phenanthroline. For example, the ECL moiety can comprise ruthenium,two bidentate bipyridyl ligands, and one substituted bidentate bipyridylligand. For example, the ECL moiety can contain a tetradentate ligandsuch as a porphyrin or substituted porphyrin.

In some embodiments, the ECL moiety can have one or more monodentateligands, a wide variety of which are known to the art. Suitablemonodentate ligands can be, for example, carbon monoxide, cyanides,isocyanides, halides, and aliphatic, aromatic and heterocyclicphosphines, amines, stibines, and arsines.

In some embodiments, one or more of the ligands of M can be attached toadditional chemical labels, such as, for example, radioactive isotopes,fluorescent components, or additional luminescent ruthenium- orosmium-containing centers.

For example, the ECL moiety can be tris(2,2′-bipyridyl)ruthenium(II)tetrakis(pentafluorophenyl)borate. For example, the ECL moiety can bebis[(4,4′-carbomethoxy)-2,2′-bipyridine]2-[3-(4-methyl-2,2′-bipyridine-4-yl)propyl]-1,3-dioxolaneruthenium (II). For example, the ECL moiety can be bis(2,2′bipyridine)[4-(butan-1-al)-4′-methyl-2,2′-bipyridine]ruthenium (II). For example,the ECL moiety can be bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine-4′-yl)-butyric acid]ruthenium (II). Forexample, the ECL moiety can be(2,2′-bipyridine)[cis-bis(1,2-diphenylphosphino)ethylene]{2-[3-(4-methyl-2,2′-bipyridine-4′-yl)propyl]-1,3-dioxolane}osmium(II). For example, the ECL moiety can be bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine)-butylamine]ruthenium (II). For example,the ECL moiety can be bis(2,2′-bipyridine)[1-bromo-4(4′-methyl-2,2′-bipyridine-4-yl)butane]ruthenium (II). Forexample, the ECL moiety can be bis(2,2′-bipyridine)maleimidohexanoicacid, 4-methyl-2,2′-bipyridine-4′-butylamide ruthenium (II).

In some embodiments of the present invention, anassay-performance-substance is used, wherein theassay-performance-substance comprises (i) an ECL moiety and (ii) alabeled binding partner for an analyte or a labeled analog of theanalyte.

In some embodiments of the present invention, theassay-performance-substance comprises an ECL moiety.

In some embodiments, the ECL moiety comprises a metal ion selected fromosmium and ruthenium.

In some embodiments, the ECL moiety comprises a derivative oftrisbipyridyl ruthenium (II) [Ru(bpy)₃ ²⁺].

In some embodiments, the ECL moiety can be [Ru(sulfo-bpy)₂bpy]²⁺ whosestructure is

wherein W is a functional group such as an NHS ester, an activatedcarboxyl, an amino group, a hydroxyl group, a carboxyl group, ahydrazide, a maleimide, or a phosphoramidite. This functional group canreact with a biological material, binding reagent, enzyme substrate orother assay reagent to form a covalent linkage.

In some embodiments, the ECL moiety does not comprise a metal. Suchnon-metal ECL moieties can be, but are not limited to, rubrene and9,10-diphenylanthracene.

C. ECL Coreactant

The tem “ECL coreactant,” as used herein, pertains to a chemicalcompound that either by itself or via its electrochemical reductionoxidation product(s), plays a role in the ECL reaction sequence. Forsimplicity, as used herein, ECL coreactants are described without regardto acid-base reactions; all acid-base forms of the stated compounds arealso contemplated and claimed.

Often ECL coreactants can permit the use of simpler means for generatingECL (e.g., the use of only half of the double-step oxidation-reductioncycle) and/or improved ECL intensity. In some embodiments, ECLcoreactants can be chemical compounds which, upon electrochemicaloxidation/reduction, yield, directly or upon further reaction, strongoxidizing or reducing species in solution. An ECL coreactant can beperoxodisulfate (i.e., S₂O₈ ²⁻, persulfate) which is irreversiblyelectro-reduced to form oxidizing SO₄.⁻ ions. The ECL coreactant canalso be oxalate (i.e., C₂O₄ ²⁻) which is irreversibly electro-oxidizedto form reducing CO₂.⁻ ions. A class of ECL coreactants that can act asreducing agents is amines or compounds containing amine groups,including, for example, tri-n-propylamine (i.e., N(CH₂CH₂CH₂)₃, TPA). Insome embodiments, tertiary amines can be better ECL coreactants thansecondary amines. In some embodiments, secondary amines can be betterECL coreactants than primary amines.

In some embodiments, the electrochemical cell of the present inventionfurther comprises an ECL coreactant.

In some embodiments, the ECL coreactant comprises a tertiary amine.

In some embodiments, the ECL coreactant comprises a tertiary aminecomprising a hydrophilic functional group.

In some embodiments, the ECL coreactant is an amine having a structureNR¹R²R³, wherein R¹, R² and R³ are C₁₋₁₀ aliphatic groups wherein atleast one of the C₁₋₁₀ aliphatic groups is substituted with at least onehydrophilic functional group. In some embodiments, the hydrophilicfunctional group is a charged group, for example, a negatively chargedgroup. Hydrophilic functional groups can be hydroxyl, hydroxycarbonyl,amino, aminocarbonyl, amidine, imino, cyano, nitro, nitrate, sulfate,sulfonate, phosphate, phosphonate, silicate, carboxylate, borate(B(OH)₃), guanidinium, carbamide, carbamate, carbonate, sulfamide,silyl, siloxy, and amide.

In some embodiments, the ECL coreactant has the structure(n-propyl)₂N(CH₂)_(n1)R*: wherein n1 is an integer from 1 to 10; and R*is a hydrophilic functional group, as defined above. In someembodiments, n1 is 2. In some embodiments, n1 is 3. In some embodiments,n1 is 4.

In some embodiments, the ECL coreactant has the formula

wherein X is selected from —(CH₂)—, —(CHR¹¹)—, —(CR¹¹R¹²)—, aheteroatom, and —N(R¹¹)—; R is a C₁₋₁₀ aliphatic group substituted withat least one hydrophilic functional group; each of R¹¹ and R¹² isindependently a C₁₋₁₀ aliphatic group optionally substituted with atleast one hydrophilic functional group; and n and m are independentlyintegers from 1 to 10.

In some embodiments, the heteroatom can be, for example, —O—or —S—.

In some embodiments, n is 2. In some embodiments, n is 3. In someembodiments, n is 4. In some embodiments, m is 2. In some embodiments, mis 3. In some embodiments, m is 4.

In some embodiments, R¹¹ is a C₁₋₄ aliphatic group.

In some embodiments, R is a C₁₋₄ aliphatic group substituted with atleast one hydrophilic functional group.

When X is —N(R¹¹)—, R¹¹ can be, for example, —(CH2)_(n3)—R¹³, wherein n3is an integer from 3 to 20, or, for example, from 3 to 10, and R¹³ is H,an aliphatic group, or a hydrophilic functional group. In someembodiments n3 is 3. In some embodiments n3 is 4.

In some embodiments, R is —(CH₂)_(n2)—R¹², wherein n2 is an integer from3 to 20, for example 3 to 10.

In some embodiments n2 is 3. In some embodiments n2 is 4. In someembodiments n2 is 5.

In some embodiments, R¹² is a hydrophilic functional group. In someembodiments R¹² is a carboxylate or sulfonate.

The use of ECL coreactants having hydrophilic functional groups (and, inparticular, ECL coreactants that are zwitterionic at neutral pH) has avariety of advantages that are unrelated to their ability to act as ECLcoreactants. These species tend to be highly water soluble and to havelow vapor pressure. For these reasons it is possible to produce highlyconcentrated stock solutions that may be diluted as necessary for use.It is also possible to prepare dried reagents comprising the ECLcoreactants without uncertainty due to loss of ECL coreactant in thevapor phase. Furthermore, when present in a dry composition, these ECLcoreactants resolubilize quickly in a minimum of volume.

ECL coreactants include, but are not limited to, lincomycin;clindamycin-2-phosphate; erythromycin; 1-methylpyrrolidone; diphenidol;atropine; trazodone; hydroflumethiazide; hydrochlorothiazide;clindamycin; tetracycline; streptomycin; gentamicin; reserpine;trimethylamine; tri-n-butylphosphine; piperidine; N,N-dimethylaniline;pheniramine; bromopheniramine; chloropheniramine; diphenylhydramine;2-dimethylaminopyridine; pyrilamine; 2-benzylaminopyridine; leucine;valine; glutamic acid; phenylalanine; alanine; arginine; histidine;cysteine; tryptophan; tyrosine; hydroxyproline; asparagine; methionine;threonine; serine; cyclothiazide; trichlormethiazide;1,3-diaminopropane; piperazine, chlorothiazide; barbituric acid;persulfate; penicillin; 1-piperidinyl ethanol; 1,4-diaminobutane;1,5-diaminopentane; 1,6-diaminohexane; ethylenediamine;benzenesulfonamide; tetramethylsulfone; ethylamine; n-hexylamine;hydrazine sulfate; glucose; n-methylacetamide; phosphonoacetic acid;and/or salts thereof

ECL coreactants include, but are not limited to, 1-ethylpiperidine;2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol (BIS-TRIS);1,3-bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane)(BIS-TRIS propane); 2-Morpholinoethanesulfonic acid (MES);3-(N-Morpholino)propanesulfonic acid (MOPS);3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO);4-(2-Hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) (HEPPSO);4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS);4-(N-Morpholino)butanesulfonic acid (MOBS);N,N-Bis(2-hydroxyethyl)glycine (BICINE); DAB-AM-16, Polypropyleniminehexadecaamine Dendrimer (DAB-AM-16); DAB-AM-32, Polypropyleniminedotriacontaamine Dendrimer (DAB-AM-32); DAB-AM-4, Polypropyleniminetetraamine Dendrimer (DAB-AM-4); DAB-AM-64, Polypropyleniminetetrahexacontaamine Dendrimer; DAB-AM-8, Polypropylenimine octaamineDendrimer (DAB-AM-8); di-ethylamine; dihydronicotinamide adeninedinucleotide (NADH); di-iso-butylamine; di-iso-propylamine;di-n-butylamine; di-n-pentylamine; di-n-propylamine; di-n-propylamine;ethylenediamine tetraacetic acid (EDTA); Glycyl-glycine (Gly-Gly);N-(2-Acetamido)iminodiacetic acid (ADA);N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES);N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid) (HEPBS);N,N-Bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (D IPSO);N,N-Bis(2-hydroxyethyl)taurine (BES); N-ethylmorpholine; oxalic acid;Piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (POPSO); s-butylamine;sparteine; t-butylamine; triethanolamine; tri-ethylamine;tri-iso-butylamine; tri-iso-propylamine; tri-n-butylamine;tri-n-butylamine; tri-n-pentylamine;N,N,N′,N′-Tetrapropyl-1,3-diaminopropane; oxalate; peroxodisulfate;piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES); tri-n-propylamine;3-dimethylamino-1-propanol; 3-dimethylamino-2-propanol;1,3-Bis(dimethylamino)-2-propanol; 1,3-Bis(diethylamino)-2-propanol;1,3-Bis(dipropylamino)-2-propanol;N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES);piperazine-N,N′-bis-3-propanesulfonic acid (PIPPS);piperazine-N,N′-bis-4-butanesulfonic acid (PIPBS);1,6-diaminohexane-N,N,N′,N′-tetraacetic acid;4-(di-n-propylamino)-butanesulfonic acid;4-[bis-(2-hydroxyethane)-amino]-butanesulfonic acid;azepane-N-(3-propanesulfonic acid); N,N-bispropyl-N-4-aminobutanesulfonic acid;piperazine-N,N′-bis-3-methylpropanoate;piperazine-N-2-hydroxyethane-N′-3-methylpropanoate;piperidine-N-(3-propanesulfonic acid); piperidine-N-(3-propionic acid)(PPA); 3-(di-n-propylamino)-propanesulfonic acid; and/or salts thereof.

In some embodiments, the ECL coreactant is selected frompiperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), tri-n-propylamine,N,N,N′,N′-Tetrapropyl-1,3-diaminopropane,1,3-Bis(dipropylamino)-2-propanol, and salts and mixtures thereof.

In some embodiments, the ECL coreactant is selected from oxalate ortri-n-propylamine.

D. Electrochemical Cells and Electrodes

The term “half-cell” refers to half of an electrolytic or voltaic cell,where either oxidation or reduction occurs.

The term “half-cell reaction” refers the reaction that occurs whenelectrons are exchanged between an electrode in the half-cell and theelectrolyte. At the anode the half-cell reaction is oxidation, while thehalf-cell reaction at the cathode is reduction.

The term “half-cell product” refers the products formed from a half-cellreaction.

The term “working electrode” refers to the test or specimen electrode inan electrochemical cell where one half-cell reaction takes place. In thepresent instance the working electrode is also a faradaic electrode.

The term “counter electrode” refers to an electrode in anelectrochemical cell to which charge flows, which charge is necessarilyof an opposite sign to that charge that flows to the working electrode.In some embodiments the counter electrode is also a capacitiveelectrode.

The term “reference electrode” refers to a nonpolarizable electrode witha known and highly reproducible potential used for potentiometric andvoltammetric analyses. The reference electrode provides a stablereference point, against which the voltage of a working electrode ismeasured. Typical reference electrodes are the silver/silver chlorideelectrode and the calomel electrode. High stability of the potential ofthe reference electrode is achieved by employing an oxidation-reductionsystem where all participants (usually two, an oxidant and reductant) ofthe reaction are present at high concentrations. During use nosignificant current is passed so as to alter the concentration ofoxidant or reductant. A reference electrode (RE) differs from a faradaicelectrode.

The term “faradaic electrode” refers to an electrode that generallyobeys Faraday's law while being used in an electrochemical cell.Faradaic electrodes exclude reference electrodes. A faradaic electrodeallows significant current to pass which can alter the concentrationsome or all of the oxidation-reduction participants. For example, afaradaic electrode may operate on a system where only the oxidant isinitially present so as to form a reductant. For example, a faradaicelectrode may operate on a system where only the reductant is initiallypresent so as to form a oxidant.

Faradaic electrodes can be made from metals and semiconductors, such asplatinum sheet electrodes, platinum wire electrodes, platinum alloyelectrodes (including alloying elements Ni, Pd, Au, Co, Fe, Ru, Os, Cr,Mo, Zr, Nb, Ir, Rh, and W), iridium electrodes, iridium alloy electrodes(including alloying elements Ni, Pd, Co, Fe, Ru, Os, Cr, Mo, Zr, Nb, Pt,Rh, and W), rhodium electrodes, rhodium alloy electrodes (includingalloying elements Au, Ni, Pd, Co, Fe, Ru, Os, Cr, Mo, Zr, Nb, Ir, Pt,and W), glassy carbon electrodes, graphite electrodes, carbonelectrodes, carbon ink electrodes, gold electrodes, silver electrodes,silver alloy electrodes, nickel electrodes, nickel alloy electrodes,stainless steel working electrodes, and the like. See, for example, Bardand Faulkner; Wiley, Electrochemical Methods: Fundamentals andApplications: 2 ed (2000).

In some embodiments of the present invention, the faradaic workingelectrode comprises carbon, gold, gold alloys, platinum, platinumalloys, iridium, iridium alloys, silver, sliver alloys, nickel, nickelalloys, stainless steel, or mercury.

In some embodiments, the faradaic working electrode is anultramicroelectrode.

The term “capacitive electrode” refers to an electrode that has limitedelectron transfer to the electrolyte solution. The capacitive electrodeis characterized by an ability to build up or trap charge within theelectrode. The charge trapped within the electrode is stabilized by thebuild-up or collection of ionic species (a so-called double layer) inthe electrolyte at the electrode surface/electrolyte interface. Anelectrode may be considered to be a capacitive electrode even if somegalvanic current is measurable.

One method to characterize a capacitive electrode is via the amount ofgalvanic current per unit area that can pass through the electrode. Thiscurrent is sometimes referred to as a “leakage current density”. For thesake of clarity, the leakage current is measured using the followingmethod. 1. Use a cell containing (a) the test electrode with geometricarea of 0.1 cm², (b) a silver/silver chloride reference electrode whoseelectrochemical area is at least 10 times the test electrode area andwherein the separation between the electrodes is less than the largestdimension of the reference electrode, and (c) an inert supportingelectrolyte: aqueous 3 M KCl, pH 6 to 8 (adjusted with HCl or KOH).Apply voltage that steps from the rest potential (the potential at whichno current flows) to the rest potential plus 0.32 V. Measure the currentas a function of time. The experiment-1-time-constant is the absolutevalue of the ratio of the current and the slope at the time at which theslope of the current decay is greatest. 2. Using same cell from step 1,add ferricyanide and ferrocyanide such that final concentration of eachis 1 mM, provide sufficient solution stirring such that the stirred cellhas a mass transfer coefficient of 10⁻² cm/s, use a total solutionvolume such that the volume is greater than or equal to the testelectrode area multiplied by mass transfer coefficient multiplied byexperiment-1-time-constant multiplied by 100, then apply same voltagewaveform as step 1. The “leakage current” is defined as the currentmeasured in this second step after 25 experiment-1-time-constants. Theleakage current density is the defined as the leakage current divided bythe test electrode area.

In some embodiments, a capacitive electrode is one whose leakage currentdensity is less than 10 μA/cm². In some embodiments, a capacitiveelectrode is one whose leakage current density is less than 1 μA/cm². Insome embodiments, a capacitive electrode is one whose leakage currentdensity is less than 100 nA/cm². In some embodiments, a capacitiveelectrode is one whose leakage current density is less than 10 nA/cm².In some embodiments, a capacitive electrode is one whose leakage currentdensity is less than 1 nA/cm². In some embodiments, a capacitiveelectrode is one whose leakage current density is less than 100 pA/cm².In some embodiments, a capacitive electrode is one whose leakage currentdensity is from about 100 pA/cm² to about 10 μA/cm². In someembodiments, a capacitive electrode is one whose leakage current densityis from about 1 nA/cm² to about 1 μA/cm².

One method to characterize a capacitive electrode is via the electrode'stime constant. The time constant is defined as the product of theelectrode resistance and electrode capacitance. The following apparatusis used to measure these quantities (which is intended to minimize theimpedance of the solution and a non-test electrode in order to betterascertain the properties of a test electrode): (1) a cell containing thetest electrode and an Ag/AgCl electrode whose electrochemical area is atleast 10 times the area of the test electrode wherein the separationbetween the electrodes is less than the largest dimension of thereference electrode; (2) an aqueous solution in the cell consistingessentially of 760 μM K₄Fe(CN)₆, 760 μM K₃Fe(CN)₆, and 1 M KCl, pH 6-8(adjusted with KOH and/or HCl); and (3) a voltage generator that appliesa DC bias voltage equal to the rest potential (the potential at whichthe current is 0) plus a 59 mV peak sinusoid and a current meter. Aswept sine technique is used to measure the frequency dependence of thecomplex impedance (applied voltage divided by measured current). Let z1equal the asymptotic value of the magnitude of the complex impedance asthe frequency approaches 0. Let z2 equal the asymptotic value of themagnitude of the complex impedance in the high frequency limit. Letω_(min) equal the radian frequency at which the phase angle of thecomplex impedance is the most negative. The electrode resistance equalsz1-z2. The electrode capacitance equals

$\frac{\sqrt{z\; {1/z}\; 2}}{\left( {{z\; 1} - {z\; 2}} \right)\; \omega_{win}}.$

In some embodiments, a capacitive electrode is one whose time constantis greater than or equal to 1 second. In some embodiments, a capacitiveelectrode is one whose time constant is greater than or equal to 10seconds. In some embodiments, a capacitive electrode is one whose timeconstant is greater than or equal to 100 seconds. In some embodiments, acapacitive electrode is one whose time constant is greater than or equalto 1,000 seconds. In some embodiments, a capacitive electrode is onewhose time constant is greater than or equal to 10,000 seconds. In someembodiments, a capacitive electrode is one whose time constant is fromabout 1 second to about 100,000 seconds. In some embodiments, acapacitive electrode is one whose time constant is from about 5 secondsto about 10,000 seconds. In some embodiments, a capacitive electrode isone whose time constant is from about 10 seconds to about 1,000 seconds.In some embodiments, a capacitive electrode is one whose time constantis from about 10 seconds to about 100 seconds.

One method to characterize a capacitive electrode is by the ratio of thetime constants of the capacitive electrode and the working electrode.This ratio is related to the ratio of the amount of electrochemicalproduct generated at the two electrodes. Typically, as the time constantof the capacitive electrode increases relative to the working electrode,the amount of electrochemical product generated at the capacitiveelectrode relative to the working electrode decreases. In thisdefinition, a capacitive electrode cannot be defined in isolation;rather it is defined relative to a particular working electrode. Tocompute the ratio of the time constants of the capacitive electrode andthe working electrode, each time constant is measured individually usingthe method described in the previous paragraph. In some embodiments, acapacitive electrode is one whose time constant is 10 times larger thanthe time constant of the working electrode. In some embodiments, acapacitive electrode is one whose time constant is 100 times larger thanthe time constant of the working electrode. In some embodiments, acapacitive electrode is one whose time constant is 1,000 times largerthan the time constant of the working electrode. In some embodiments, acapacitive electrode is one whose time constant is 10,000 times largerthan the time constant of the working electrode.

In some embodiments, the capacitive counter electrode is an ideallypolarized electrode.

In some embodiments, the capacitive counter electrode comprises asemiconductive material with an oxide layer.

In some embodiments, the capacitive counter electrode comprises aconductive material with an oxide layer.

In some embodiments, the capacitive counter electrode comprises amaterial whose bulk resistivity is less than about 10⁻² Ωm at 20° C.,covered by an electrically insulating material having a bulk resistivitygreater than 10⁴ Ωm at 20° C. wherein the insulating material has a meanthickness that is less than about 1 μm.

In some embodiments, the insulating material has a bulk resistivitygreater than 10⁵ Ωm at about 20° C. and the insulating material has amean thickness that is less than about 100 nm.

In some embodiments, the capacitive counter electrode comprises oxidesof silicon, titanium, aluminum, magnesium, zirconium, and/or tantalum.

The term “electrolyte” refers to a medium that provides the iontransport mechanism between the electrodes of an electrochemical cell.Generally electrolytes are substances that dissociates into ions when insolution and are capable of conducting electricity, such as acids,bases, and salts.

The term “capacitance” refers to that property which permits the storageof electrically separated charges when potential differences existbetween conductors. The capacitance of a two-terminal capacitor isdefined as the ratio between the electric charge ohmically connected toone terminal and the resultant difference in potential between theterminals. In the present invention the capacitive electrode acts like acapacitor when it stores charge within the electrode and collectscounter charges at the electrode electrolyte interface.

The term “permittivity” equals to the electric flux density (D) dividedby the electric field strength (E). The permittivity of a material canchange with frequency. In these electrochemical applications, therelevant frequency range is low, with the upper end rarely exceeding10's of kHz. The capacitance of a parallel plate capacitor isapproximately equal to the permittivity of the material between theplates multiplied by the area of one of the plates divided by distancebetween the plates.

“Coulometric methods” are known in the art and can be used toquantitatively add electrons to or remove electrons from a system.Coulometric methods involve electrochemical generation of chemicalspecies in a solution via an electrode reaction. The amount of product(N_(o)) generated at the faradaic working electrode can be controlled bythe charge passed at that electrode, as governed by Faraday's law:N_(o)=q/(nF) where q is the charge passing through the electrode, n isthe number of electrons used to generate 1 molecule of product at theworking electrode, and F is Faraday's constant. When using coulometricmethods, care must be taken to account for the electrode's capacitance.

Capacitive electrodes can be made from oxidized metals andsemiconductors. Further examples of capacitive electrodes includeelectrodes made from materials having a bulk resistivity less than 10⁻²Ω/m at 20° C., covered by a thin (mean thickness less than 10 μm)insulating material, including dielectrics for integrated circuitfabrication such as silicon dioxide, silicon nitride, TiN, TaN, TiAlN,TaAlN, TaSiN, SrTiO₃, Ta₂O₅, TiO₂, Y₂O₃, ZrO₂, HfO₂, Al₂O₃, BaSrTiO₃,FOx®-1x and FOx®-2x (Dow Corning, Midland, Mich.) flowable oxide productfamilies (comprising hydrogen silsesquioxane); solder masks (asdescribed in the standard IPC-SM-840C: Qualification and performance ofpermanent solder mask); and conformal coatings (as described in thestand IPC-SM-840 Qualification and Performance of Permanent Solder Mask)such as parylene and Novec™ EGC-1700, EGC-1702, EGC-1704, and EGC-1720electronic coatings (3M, MN).

Further examples of capacitive electrodes include electrodes made frommaterials having a bulk resistivity less than 10⁻² Ωm at 20° C., coveredby a thin (mean thickness less than 1 μm) insulating material such asoxides of silicon, titanium, aluminum, magnesium, zirconium, and/ortantalum.

Further examples of capacitive electrodes include electrodes made frommaterials having a bulk resistivity less than 10⁻² Ωm at 20° C., coveredby a thin (mean thickness less than 1 μm) insulating material depositedby chemical vapor deposition or by spin coating.

Further examples of capacitive electrodes include electrodes made frommaterials having a bulk resistivity less than 10⁻² Ωm at 20° C., coveredby a thin (mean thickness less than 1 μm) insulating material having abulk resistivity greater than 10⁴ Ωm at 20° C. Thinner insulating layerscan provide increased capacitance for a given electrode area; however,thinner insulating layers may cause increased leakage currents throughthe presence of pin-holes in the insulation or by dielectric breakdown.In some embodiments, the mean thickness of the insulating layer on thecapacitive electrode is less than 10 μm. In some embodiments, the meanthickness of the insulating layer on the capacitive electrode is lessthan 1 μm. In some embodiments, the mean thickness of the insulatinglayer on the capacitive electrode is less than 100 nm. In someembodiments, the mean thickness of the insulating layer on thecapacitive electrode is less than 10 nm. In some embodiments, the meanthickness of the insulating layer on the capacitive electrode is lessthan 1 nm. In some embodiments, the mean thickness of the insulatinglayer on the capacitive electrode is from about 1 nm to about 10 μm. Insome embodiments, the mean thickness of the insulating layer on thecapacitive electrode is from about 10 nm to about 1 μm. In someembodiments, the mean thickness of the insulating layer on thecapacitive electrode is from about 10 nm to about 100 nm.

The capacitance of a capacitive electrode increases with thepermittivity of the insulating layer. In some embodiments, theinsulating layer has a permittivity that is about equal to thepermittivity of free space. In some embodiments, the insulating layerhas a permittivity that is greater than about 1 times and less thanabout 3 times the permittivity of free space. In some embodiments, theinsulating layer has a permittivity that is greater than about 3 timesand less than about 5 times the permittivity of free space. In someembodiments, the insulating layer has a permittivity that is greaterthan about 5 times and less than about 10 times the permittivity of freespace. In some embodiments, the insulating layer has a permittivity thatis greater than about 10 times and less than about 100 times thepermittivity of free space. In some embodiments, the insulating layerhas a permittivity that is greater than about 100 times the permittivityof free space.

II. Certain Embodiments of the Cell and Electrodes

The present invention is directed to methods and apparatus utilizing anelectrochemical cell comprising a faradaic working electrode and acapacitive counter electrode. The capacitive counter electrode can becharacterized by, for example, its leakage current density, its timeconstant, and the ratio of its time constant to the working electrode'stime constant, or combinations thereof. In some embodiments theelectrochemical apparatus is operated in a manner that allows a constantfaradaic current at the working electrode while producing little to nooxidation or reduction products at the counter electrode. In someembodiments the electrochemical apparatus is operated in a manner thatallows a constant faradaic current at the working electrode whilereducing the amount of oxidation or reduction products at the counterelectrode.

Typical electrochemical cells employ faradaic electrodes at both theanode (where oxidations occur) and the cathode (where reductions occur).A separate half-reaction occurs at each of these electrodes, producing adifferent product (half-cell product) at each of the electrodes. Thesolution as a whole maintains charge (ionic) neutrality at all timesbecause equal charges of anionic and cationic species are generated byeach of the half-cell reactions at the corresponding electrodes.Generally only the reaction or product at the working electrode is ofinterest. In fact, the counter electrode is often placed in a differentcompartment, separated by an ionically conducting “cell-separator” toprevent mixing of the half-cell products formed at each of theelectrodes. Salt bridges and other ionically conducting cell-separatorsare known in the art. See, for example, Bard and Faulkner; Wiley,Electrochemical Methods: Fundamentals and Applications: 2 ed (2000).

The present invention can be used in many sizes of electrochemical cellsthat comprise a faradaic working electrode and a capacitive counterelectrode. Exemplary size ranges for the faradaic working electrodeinclude macroscopic geometrical areas of about 0.1 μm² to about 10 m²,about 10 μm² to about 10000 cm², about 10 μm² to about 100 cm², about10000 μm² to about 10 cm², about 1 mm² to about 100 mm², and about 1 mm²to about 10 mm². Exemplary sizes for the faradaic working electrodeinclude macroscopic geometrical areas of about 0.1 μm², 1 μm², 10 μm²,100 m², 1000 μm², 10000 μm, 1 mm², 10 mm², 100 mm², 1 cm², 10 cm², 100cm², 1000 cm², 10000 cm², 1 m², or 10 m². Exemplary sizes and sizeranges the capacitive counter electrode include those sizes for thefaradaic working electrode plus larger sizes to accommodate suchembodiments as electrode 202.

In some embodiments, the electrochemical cell can partially enclose asample volume. For example, the cell can be a well in a multi-well assayplate, a beaker, a tube, a flow cell, or other shapes and sizes. In someembodiments, the faradaic and the capacitive counter electrode can be ina single cell, for example cell 200. In some embodiments, the counterelectrode and the working electrode can be in individual cellsconductively connected to one another by a salt bridge, frit, or otherbridging means, which are well known in the art. The cell can also beconfigured so that only one of the faradaic working electrode and thecounter electrode is positioned within the cell, and the other ispositioned adjacent to the cell, but in contact with the fluid withinthe cell. In addition, the cell can be one of a plurality of single orsplit cells, for example, the wells of a multi-well plate or multipletubes in an auto-sampling system.

The electrochemical cell can also be a flow cell. The flow cell can beconfigured with an inlet and outlet such that the fluids can flowthrough the inlet into the cell and then flow out of the cell throughthe outlet. Exemplary electrochemiluminescence flow cells and methodsfor their use are disclosed in U.S. Pat. No. 6,200,531.

In some embodiments, the electrochemical cell is capable of receiving anelectrolyte solution. This solution can participate in electrochemicalreactions at the faradaic working electrode when contacted by thefaradaic working electrode and the capacitive counter electrode in theelectrochemical cell and electrical energy is applied between theelectrodes.

In some embodiments, the electrochemical cell can further comprise areference electrode. In some embodiments, the cell does not comprise areference electrode. The reference electrode, when present, can be usedto better control the potential drop across the faradaic workingelectrode irrespective of the potential drop across the capacitivecounter electrode until the voltage compliance limit of the potentiostatis attained or dielectric breakdown occurs at the capacitive counterelectrode.

III. Certain Embodiments of the Apparatus

In some embodiments, the apparatus can comprise anassay-performance-substance comprising at least one of (a) a labeledbinding partner for an analyte and (b) a labeled analog of the analyte.In some embodiments, the assay-performance-substance can be a drycomposition. In some embodiments, the assay-performance-substancecomprises an ECL moiety. In these embodiments, the apparatus mayoptionally comprise an ECL coreactant.

In some embodiments, the apparatus further comprises a vapor barrierthat encloses the assays-performance-substance, for example, to preventevaporation of a liquid composition or to prevent melt-back of a drycomposition.

In some embodiments, the apparatus can comprise a filter in fluidicconnection to the faradaic working electrode and capacitive counterelectrode. Filtration is one method for removing interfering componentsof the sample matrix. The interfering components can be removed from thesample matrix prior to the sample entering the electrochemical cell. Inthis approach, the barrier of the filter can retain particulatecomponents that might interfere with the detection process. Particulatecomponents of the matrix can, for example, interfere with some of thedetection methodologies that require capture or deposition of beads on adetection surface. Additionally, removing interfering components candecrease the amount of surface that is available to bind analyte. Insome embodiments, the filter entrapped particulate components can betreated to release analyte for the detection process.

Filtration primarily separates components from solutions by presenting aphysical barrier that can exclude particles larger than a given size.There are many different methods in the filtration art to make barriersof these types, each method a function of the base material beingmanipulated. For example, metal wire is commonly used to make wovenscreens that can be used to catch extremely large particles, forexamples, particles over 50 microns in size. To capture smaller airborneparticles, smaller diameter metal wire screens can be used, but theyhave limitations due to impedance to air flow (pressure drop).Polymer-based membranes are typically used to remove smaller particlesfrom solutions. For example, the polymer nylon can be used in a phaseinversion casting process to make membranes that range from about a 10micron pore size rating, down to about a 0.1 micron pore size rating.Other polymer-based membranes (e.g. polyethersulphone, nitrocellulose,or cellulose acetate) can be made by a solvent evaporation castingprocess. The filtration medium is generally incorporated into a holdingdevice that allows the fluid of interest to pass through the filterbarrier in a controlled manner. In some embodiments, the invention canuse a filter-containing filtration media using the polymerpolyethersulphone (PES). In certain embodiments, the PES filter can beencased in a plastic housing that can be (a) attached to a syringe, (b)part of a single use disposable designed to ease robotic automation, or(c) part of a multiple-use disposable designed to filter a plurality ofsamples.

Filtration primarily separates components from solutions by size. Inmost filters, the pathway through the filter is not a straight hole, butrather a twisted path. This makes describing filter hole size somewhatoperational in nature, and gives rise to the term “Pore Size Rating”. Indetermining a filter's Pore Size Rating, filters are challenged with aknown volume (or amount) of particles of a known size (known by asecondary means like microscopy, light scattering, or impedancemeasurements). Then, the amount of particles downstream of the filter ismeasured and compared to the amount of particles upstream of the filter,across multiple sizes of particles. When the ratio of downstream toupstream particles drops significantly below unity for a given sizerange of particles, the filter is said to have removal capacity for thatsize range. These ratios are typically described in logarithmic-basedunits of removal. For example, a filter rated at 5 microns willtypically reduce the level of downstream particles greater than 5microns by a ratio of 0.90 (90% removal or 1 log removal) to a ratio of0.999 (99.9% removal: or 3 log removal). The pore size of the filter canbe chosen based many factors. In some embodiments, the pore size can belarge enough to pass the analyte, for example, anthrax spores, which areapproximately 1 μm in size. In certain embodiments, the pore size can besmall enough to block interfering components from the sample matrix. Thepore size can also affect the rate of fluid flow across the filter, withsmaller pores generally creating larger resistances to flow.

In some embodiments, the filter has a pore size rating of 5 microns. Insome embodiments, the filter has a pore size rating of 0.1, 0.2, 0.5, 1,2, 3, 4, 7, 10, 15, 20, 50, or 100 microns. In some embodiments, thefilter has a pore size rating less than or equal to about 100 micronsand greater than or equal to about 10 microns. In some embodiments, thefilter has a pore size rating less than or equal to about 10 microns andgreater than or equal to about 1 microns. In some embodiments, thefilter has a pore size rating less than or equal to about 1 microns andgreater than or equal to about 0.1 microns. In some embodiments, thefilter has a pore size rating less than or equal to about 0.1 micronsand greater than or equal to about 0.02 microns.

In some embodiments, the apparatus comprises (a) an electrochemical cellcomprising a faradaic working electrode and a capacitive counterelectrode wherein the electrochemical cell is capable of receiving anelectrolyte solution; and (b) any combination of the referenceelectrode, an assay-performance-substance comprising at least one of (i)a labeled binding partner for an analyte and (ii) a labeled analog ofthe analyte, the ECL coreactant, and the filter. In certain embodiments,the apparatus is an expendable or replaceable element in a largerinstrument, for example, to prevent the larger instrument fromcontacting the electrolyte solution (which may contain a samplecomprising an analyte that is desired to be detected). In certain ofthese embodiments, the apparatus comprises theassay-performance-substance in a dry composition and a vapor barrierthat encloses the assay-performance-substance.

In some embodiments, the apparatus comprises any of the aboveembodiments (for example, an apparatus comprising an electrochemicalcell comprising a faradaic working electrode and a capacitive counterelectrode wherein the electrochemical cell is capable of receiving anelectrolyte solution that can simultaneously contact said faradaicworking electrode and said capacitive counter electrode), plus a sourceof electrical energy capable of being electrically connectable to thefaradaic working electrode and the capacitive counter electrode.Consistent with the principles of the invention, there are many possiblesources of electrical energy. The electrical energy applied to theelectrochemical cell can be generated using conventional instrumentationsuch as potentiostats, galvanostats, and/or similar instruments. In someembodiments, a voltage source, a current source, a source of charge,and/or a power source can be used with either a constant or atime-varying waveform comprising offsets, steps, ramps, sinusoids, andcombinations thereof. In some embodiments, a third electrode can be usedas a feedback mechanism to control the voltage across the faradaicworking electrode. In some embodiments, the energy can come from a localenergy store, such as a battery, super-capacitor, flywheel, and/or ahydrocarbon source (e.g., gasoline and/or methane). In some embodiments,a photovoltaic device can provide the energy. In some embodiments, powerfrom the AC network can be used. In some embodiments, semiconductordevices can use the energy store to control charge, voltage, and/orcurrent applied to the electrodes over time. In some embodiments, acomputing device (e.g., a microprocessor, microcontroller, or computer)is used to control the application of electrical energy to theelectrochemical cell.

In some embodiments, the apparatus comprises any of the aboveembodiments (for example, an apparatus comprising 1. an electrochemicalcell comprising a faradaic working electrode and a capacitive counterelectrode wherein the electrochemical cell is capable of receiving anelectrolyte solution that can simultaneously contact said faradaicworking electrode and said capacitive counter electrode and 2. a sourceof electrical energy capable of being electrically connectable to thefaradaic working electrode and the capacitive counter electrode), plus amagnet located so as to be able to exert a magnetic force onmagnetizable particles located at the faradaic working electrode.

In some embodiments, the magnet can be reversibly moved from itsposition beneath the faradaic electrode to reduce the magnetic forceapplied on magnetizable beads in the sample by the magnet.

In some embodiments, the magnet can comprise either a permanent magnetor an electromagnet.

In some embodiments, the magnet can comprise a dipole magnet, a sandwichmagnet, or a channel magnet.

In some embodiments, the apparatus comprises any of the aboveembodiments (for example, an apparatus comprising 1. an electrochemicalcell comprising a faradaic working electrode and a capacitive counterelectrode wherein the electrochemical cell is capable of receiving anelectrolyte solution that can simultaneously contact said faradaicworking electrode and said capacitive counter electrode and 2. a sourceof electrical energy capable of being electrically connectable to thefaradaic working electrode and the capacitive counter electrode), plus aphotodetector positioned to detect light emitted on the faradaic workingelectrode. In some embodiments, the photodetector can be aphotomultiplier tube, photodiode, CMOS device, or a charge-coupleddevice.

In some embodiments, the apparatus comprises any of the aboveembodiments (for example, an apparatus comprising 1. an electrochemicalcell comprising a faradaic working electrode and a capacitive counterelectrode wherein the electrochemical cell is capable of receiving anelectrolyte solution that can simultaneously contact said faradaicworking electrode and said capacitive counter electrode and 2. a sourceof electrical energy capable of being electrically connectable to thefaradaic working electrode and the capacitive counter electrode), plus apump arranged to be able to move liquid across or onto the faradaicworking electrode. In some embodiments, the pump can be modeled as apressure source (e.g., an impeller pump, one that uses gravity, or apump based on capillary action), or as a volume velocity source (e.g., aperistaltic pump, syringe pump, gear pump, or positive displacementpump). In some embodiments, the pump moves liquid across the faradaicworking electrode. In some embodiments, the pump is arranged to be ableto move liquid across or onto the faradaic working electrode by usingpositive gauge pressures. In some embodiments, the pump is arranged tobe able to move liquid across or onto the faradaic working electrode byusing negative gauge pressures. In some embodiments comprising a filter,the pump can be used to remove filtrate from the filter; for example,fluid can be moved through the filter by gravity flow or by pressure, inwhich positive gauge pressure is applied upstream of the filter ornegative gauge pressure (vacuum) is applied downstream of the filter.This filtrate can, for example, be dispensed into the electrochemicalcell and onto the faradaic working electrode. In some embodimentscomprising a flow cell, the pump can be used to transport fluids intoand out of the flow cell.

The electrochemical cell can be used in conjunction with variousautomation systems such as an automatic drive mechanism or an alignmentdevice to cause relative motion between the cell and the electrodes. Anexemplary apparatus that comprises a flow-cell comprising a flow cellcomprising capacitive counter electrode and a faradaic working electrodecan further comprise a pump for aspirating and/or dispensing fluids intoand out of the flow cell and an electrical energy source to drive theelectrodes. In some embodiments, the exemplary apparatus can furthercomprise a fluid handling station for introducing one or more reagentsand/or one or more samples that can include gases and liquids. The fluidhandling station can comprise flow control valves as well as a manifoldfor accepting pipettor for aspirating/dispensing fluids from one or morelocations into the cell. Additional flow control valves can also bepresent, as well as reagent/gas detectors.

When a potential is applied to an electrochemical cell with a faradaicworking electrode and a capacitive counter electrode, electrons crossthe faradaic electrode interface and a charge builds up in thecapacitive electrode. The charge build-up in the counter electrode canbe described by the equation for a capacitor in an electrical circuit:q/V=C, where q is the charge on the capacitor (in coulombs), V is thepotential across the capacitor (in volts) and C is the capacitance (inFarads). The capacitance of an electrode may change as a function of thepotential applied to the system; for example, the distance between thedouble layer and the electrode may increase as the amount of charge inthe double layer increases. To a good approximation, the capacitivecapability of an electrode is proportional to the surface area exposedto the solution. This electrochemical surface area can be larger thanthe macroscopic geometrical surface area by roughening the surface. Theratio of the electrochemical surface area to the macroscopic geometricalsurface area can be, for example, from about 1 to about 1000, or fromabout 1 to about 10, or from about 10 to about 100, or from about 100 toabout 1000. The ratio of the electrochemical surface area to themacroscopic geometrical surface area can be, for example, greater thanor equal to about 1, about 2, about 3, about 5, about 10, about 30,about 100, about 300 or about 1,000. Methods to increase the roughnessand thereby increase the electrochemical surface area include mechanicalabrasion (e.g., through the use of sandpaper and/or sand blasting),plasma etching, ion and/or electron beam irradiation, depositing a roughcoating (e.g., using techniques such as thermal spray, plating, orelectrodeposition, ink-jet, sputtering, chemical vapor deposition),chemical etching, laser ablation, and electrochemical surfacemodification. Other methods to obtain a large roughness include using ahighly rough starting material as the electrode, for example, sinteredparticles, platinum black, carbon nanotubes and carbon black inks. Toefficiently add to the electrochemical surface area, the characteristicdimension of the roughness is preferably larger than (but not requiredto be) about 1 nm so that ions can easily contact all of the area.

When employing a voltage source to drive a capacitive counter electrodeand a faradaic working electrode, the current decreases with time ascharge is trapped in the counter electrode (See FIG. 2). One mightexpect that the amount of current at the working electrode is limited bythe amount of charge that can be contained in the counter electrode atthe applied voltage during operation of the electrochemical cell, whichis determined by the capacitance of the electrode; however, thisinvention contemplates several methods of generating additional current,if additional current is desired.

In some embodiments, the apparatus comprises any of the aboveembodiments, plus a mechanism to contact differing capacitive counterelectrodes or differing areas of the same capacitive counter electrodesto an electrolyte solution that is contacting the faradaic workingelectrode. By exposing fresh parts of the counter electrode, thecapacity increases thereby increasing the amount of current that can bedriven through the working electrode. In some embodiments, the parts ofthe counter electrode that are charged can be unused for the remainderof the useful life of the cell. In some embodiments, the parts of thecounter electrode that are charged can be discharged through contactwith the air, through contact with another solution in a different cell,through the electrode's leakage current, or by reversing the directionof the current. By continuously exposing fresh parts of the counterelectrode, faradaic currents at the working electrode can bemass-transport limited by the reactants at the working electrode.

FIG. 6 shows an exemplary embodiment of a cell whose counter electrodearea exposed to the solution changes with time. The faradaic workingelectrode (201) can remain stationary in the cell while the capacitivecounter electrode can be continuously moved through the electrolytesolution. For example, a conductive wire or film coated with a thininsulating layer (202) can be passed through the electrolyte (204) undera fixed bias as shown in FIG. 6. Anions (203) can be removed from thesolution when a negative bias is applied to the metal electrode andexcess cations can be left in the solution to provide electroneutralityfor the cathodic faradaic reaction. Similarly, cations can be removed atthe capacitive counter electrode when the working electrode is an anode.Alternatively, a streaming mercury electrode with a surface that iscontinuously renewed can be used if operated in the potential regionwhere it behaves as an ideally polarized electrode. See, for details, I.M. Kolthoff, J. J. Lingane, Polarograghy, 2nd Ed, Vol. 1, IntersciencePublishers, New York, p. 357, 1952. A Capacitive counter electrode canremove ions of a single polarity (i.e., without a counter ion) where theionic charge can be compensated by electronic charge.

Exemplary combinations of the apparatus components described aboveinclude an electrochemical cell comprising: a faradaic workingelectrode; a capacitive counter electrode; and a source of electricalenergy. In certain embodiments, the present invention is directed to anapparatus comprising an electrochemical cell comprising: a cell capableof receiving an electrolyte solution, a faradaic working electrode; anda capacitive counter electrode. In some embodiments, the cell is a flowcell.

Exemplary combinations of the apparatus components described aboveinclude an apparatus comprising an electrochemical cell used forperforming a binding assay for an analyte in a sample based uponmeasurement of electrochemiluminescence at a faradaic working electrodesurface, the electrochemical cell comprising: a cell for receiving asolution comprising (a) sample comprising the analyte and (b) a labeledbinding partner for the analyte wherein the labeled binding partnercomprises an ECL moiety; a faradaic working electrode having anelectrode surface exposed to and positioned adjacent to a portion of thesample containing volume; a capacitive counter electrode having theelectrode surface exposed to and positioned adjacent to a portion of thesample containing volume; a source of electrical energy sufficient togenerate luminescence; a magnet for collecting beads along the faradaicworking electrode surface; and a photodetector for measuring the amountof luminescence generated and, thereby, the quantity of analyte in thesample.

Exemplary combinations of the apparatus components described aboveinclude an apparatus comprising an electrochemical cell for use inanalyzing a sample suspected of containing one or more analytescomprising: a filter fluidically connected to a sampling device; a pumparranged to be able to drive liquid flow across the filter; a cell forreceiving a filtered sample suspected of containing one or moreanalytes; at least one faradaic working electrode; and at least onecapacitive counter electrode; and means for measuring the quantity ofanalyte in the sample.

IV. Exemplary Methods

The invention also contemplates methods of use, for example, ofapparatus embodiments described above. These methods of use includeassay methods for determining the presence or amount of one or moreanalytes in a sample and methods of generating at least oneelectrochemical product at a working electrode while generating adiscordantly smaller amount of electrochemical byproduct at a counterelectrode.

In some embodiments, the method for determining the presence or amountof an analyte in a sample comprises the steps of contacting both afaradaic working electrode and a capacitive counter electrode with asolution comprising the sample and an electrolyte; supplying electricalenergy between the faradaic working electrode and the capacitive counterelectrode sufficient to provide for faradaic charge transfer at thefaradaic working electrode; measuring at least one of (i) light, (ii)current, (iii) voltage, and (iv) charge to determine the presence oramount of the analyte in the sample.

In some embodiments, the method for determining the presence or amountof an analyte in a sample comprises the steps of

(a) optionally preprocessing the sample;

(b) contacting a faradaic working electrode to a solution comprising theoptionally pre-processed sample; and an electrolyte;

(c) contacting a capacitive counter electrode to the solution;

(d) supplying electrical energy between the faradaic working electrodeand the capacitive counter electrode sufficient to provide for faradaiccharge transfer at the faradaic working electrode;

(e) measuring at least one of (i) light, (ii) current, (iii) voltage,and (iv) charge to determine the presence or amount of the analyte inthe sample.

Exemplary methods utilizing the measurement of light includeluminescence assays, including fluorescence, chemiluminescence, andelectrochemiluminescence assays. Exemplary methods utilizing themeasurement of current include measuring the rate of generation of anelectrochemical reactive species in the solution that is consumed by thefaradaic working electrode. Exemplary methods utilizing the measurementof voltage include ion-selective electrodes. Exemplary methods utilizingthe measurement of charge include measuring the capacitance of thecapacitive counter electrode from which, for example, the concentrationof the dominate ion and/or the Debye length of the solution can beestimated. Exemplary methods utilizing a combination of measurementsinclude cyclic voltammograms involving measuring both current andvoltage to examine, e.g., oxidization and reduction behavior in thesolution. Various properties or combinations of properties of thephysical measurements of light, current, voltage, and/or charge can beused to determine the quantity of analyte in the sample; for example,wavelength, frequency, energy, intensity, polarity, polarization,amplitude, waveform shape, and/or time dependency can be used.

In some embodiments, the solution used in the determination of thepresence or amount of an analyte in a sample further comprises an ECLmoiety, for example, those ECL moieties that comprise ruthenium orosmium. In some embodiments, the solution further comprises an ECLcoreactant, for example, tertiary amines with and without hydrophilicfunctional groups, tertiary amines with alkyl groups optionallycomprising hydrophilic functional groups, biological buffers withtertiary amines, and/or oxalate. In some embodiments, the electricalenergy supplied between the faradaic working electrode and thecapacitive counter electrode is sufficient to induceelectrochemiluminesce and the quantity of analyte in the sample isdetermined by measuring luminescence. In some embodiments, the solutionfurther comprises at least one of a labeled binding partner for ananalyte and a labeled analog of the analyte. In some embodiments, thesolution further comprises a second binding partner for the analytewherein the second binding partner is linked to a support. In someembodiments, the support is a magnetizable bead.

In some embodiments, the method for determining the presence or amountof an analyte in a sample comprises the step of pre-processing thesample. Pre-processing can be, for example, filtering the sample througha filter to form a filtrate. Exemplary pore size ratings of the filterinclude those falling in the ranges of about 10 μm to about 100 μm,about 1 μm to about 10 μm, about 0.1 μm to about 1 μm, and about 0.02 μmto about 0.1 μm. Other pre-processing steps can be, for example, mixingthe sample with a reagent whose pH is greater than 8 or less than 6 andan osmolarity is greater than or equal to 0.1 osmol/L. Otherpre-processing steps can be, for example, mixing the sample with areagent whose osmolarity is greater than or equal to 1.1 osmol/L.

In some embodiments, the assay methods can comprise the steps ofinducing an ECL moiety in solution to emit light by applying anelectrochemiluminescence-inducing electrical waveform to the workingelectrode in the presence of the ECL moiety; and measuring theluminescence emitted by the ECL moiety. In some embodiments, the assaymethods can comprise the step of adding a second binding partner linkedto a support (e.g., a magnetizable bead). In some embodiments usingmagnetizable beads, the method can comprise the steps of magneticallycollecting the magnetizable beads along the faradaic working electrodesurface through the use of a magnet (e.g., a permanent magnet or anelectromagnet) that can optionally be reversibly removed from itslocation beneath the faradaic working electrode.

In some embodiments, the assay methods can employ a faradaic currentflow through the faradaic working electrode that alternates in directionto reduce the potential across the capacitive counter electrode.

The present invention can be used for coulometric processes, forexample, in micro and nanoscale electrochemical cells. In someembodiments, the present invention can be used to reduce the generationof an undesirable or destructive product at the counter electrode in anelectrochemical cell. In some embodiments, the present invention can beused in electrochemiluminescent assays where the amount of an analyte ina sample can be determined by the amount of light generated at theworking electrode, for example, in nanoscaled ECL assays whereelectrochemical products generated at the counter electrode havesufficient time to diffuse near the working electrode and may interferewith the assay.

The samples that can be analyzed using an electrochemical cell of thepresent invention can comprise at least one analyte. Measurement of ananalyte in the sample can be carried out by any of the numeroustechniques available in the art of biological assays, including but notlimited to, nucleic acid hybridization assays, nucleic acidamplification assays, cell culture-based assays, agglutination tests,immunoassays (or other assay formats based on the use of specificbinding partners of the marker of interest), immunochromatographicassays, enzymatic assays, etc. The detection method can be a bindingassay, such as an immunoassay, and the detection can be performed bycontacting an assay composition with one or more binding partners of theanalyte. In certain embodiments, the assay uses a sandwich orcompetitive binding assay format. Examples of sandwich immunoassaysperformed on test strips are described by U.S. Pat. No. 4,168,146 toGrubb et al. and U.S. Pat. No. 4,366,241 to Tom et al. Examples ofcompetitive immunoassay devices suitable for use with the presentinvention include those disclosed by U.S. Pat. No. 4,235,601 to Deutschet al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535to Buechler et al. In certain embodiments, at least one of the bindingpartners employed in such an assay is immobilized on a support. In someembodiments, a labeled binding partner and/or a labeled analog of theanalyte is used in the binding reactions. In some embodiments, thelabeled binding partner comprises an ECL moiety.

A binding partner can be immobilized on the support by any conventionalmeans, e.g., adsorption, absorption, noncovalent binding, covalentbinding with a crosslinking agent, or covalent linkage resulting fromchemical activation of either or both of the support or the bindingpartner. In some embodiments, the immobilization of the binding partnerby the support can be accomplished using a binding pair. For example,one member of the binding pair, e.g., streptavidin or avidin, can bebound to the support and the other member of the same binding pair,e.g., biotin, can be bound to the first binding partner. Suitable meansfor immobilizing a binding partner on the support are disclosed, forexample, in the Pierce Catalog, Pierce Chemical Company, P.O. Box 117,Rockford, Ill. 61105, 1994.

A. Certain Assay Methods

Binding assays can be carried out using magnetizable beads as a supportfor a solid phase binding assay using a flow cell-based design withpermanent reusable flow cells, or single use replaceable cells.Complexes comprising an ECL moiety that are bound to magnetizable beadscan be collected on an electrode in the flow cell with the aid of amagnet, for example, a dipole magnet, a sandwich magnet, a channelmagnet and/or an electromagnet. The labels on the collected beads can beinduced to emit ECL by application of a potential to the electrodes andthe ECL can be measured to measure the amount of label. The ECL assaymethod can also comprise the step of introducing an ECL coreactant priorto application of the ECL-inducing potential.

In certain embodiments, a solid phase sandwich immunoassay can be runusing the electrochemical cell of this invention. Two antibodiesdirected against the analyte are used: i) a capture antibody that islinked or capable of being linked (e.g., through the formation of aspecific binding pair such as a biotin-streptavidin interaction) to asolid phase and ii) a detection antibody that is linked or capable ofbeing linked (e.g., through the formation of a specific binding pairsuch as a biotin-streptavidin interaction) to a label, for example anECL moiety. A sample comprising the solubilized analyte can be contactedwith the two antibodies and the solid phase so that in the presence ofthe analyte the two antibodies can bind to the analyte to form a“sandwich complex” on the solid phase comprising the label. The label onthe solid phase can be measured so as to measure the analyte in thesample.

The sample can be introduced into the cell as-collected or the samplecan under go one or more preparations steps. See for example, THEIMMUNOASSAY HANDBOOK, 3.sup.rd edition, David Wild editor, Elsevier,2005.

In certain embodiments, the present invention can provide a method ofdetecting an analyte in a sample comprising the steps of: providing acell for receiving an electrolyte solution and the sample which maycomprise the analyte; placing the electrolyte solution in the cell;placing the sample in the cell; contacting a faradaic working electrodewith the sample and electrolyte solution; contacting a capacitivecounter electrode with the sample and electrolyte solution; supplyingelectrical energy to the electrodes sufficient to provide for faradaiccharge transfer at the faradaic working electrode that can generate anelectrochemical product; and determining the quantity of analyte in thesample.

In certain embodiments, the present invention can provide a method fordetermining the presence or amount of an analyte in a sample comprisingthe steps of (1) forming a solution comprising (a) the sample, (b) alabeled binding partner specific for said analyte wherein the label isan ECL moiety, (c) a binding partner specific for said analyte linked toa magnetizable bead, (d) an ECL coreactant, and (e) an electrolyte; (2)contacting a faradaic working electrode and a capacitive counterelectrode with the solution; (3) collecting the beads along the faradaicworking electrode; (4) supplying electrical energy to the electrodes tocause the ECL moiety to repeatedly generate electrochemiluminescence;(5) measuring said electrochemiluminescence; and (6) determiningpresence or amount of said analyte from the measurement.

In some embodiments, the method can be a method for analyzing the samplefor multiple analytes. For example, an array of faradaic workingelectrodes can be used with one or more capacitive counter electrodeswherein a measurement from one or more faradaic working electrodes isused to measure each analyte. For example, one or more faradaic workingelectrodes can be used with one or more capacitive counter electrodeswherein more than one analyte is measured on each faradaic workingelectrode.

In some embodiments, the method of generating at least oneelectrochemical product at a working electrode while generating adiscordantly smaller amount of electrochemical byproduct at a counterelectrode, comprises contacting a faradaic working electrode and acapacitive counter electrode with an electrolyte solution; and applyingelectrical energy between the faradaic working electrode and thecapacitive counter electrode wherein the faradaic charge transferredacross the faradaic working electrode is greater than the faradaiccharge transferred across the capacitive counter electrode.

The discord in the amount of byproduct formed, relative to the amount ofproduct formed, is primarily a result of the discord in faradaic chargetransfer at the working and counter electrodes. In an apparatus of thepresent invention, and unlike traditional electrochemical apparatus, theamount of charge transferred at the faradaic working electrode isgreater than the amount of charge that is transferred at the capacitivecounter electrode. The difference in the amount of charge transferred atthe working and counter electrodes can be, for example, greater than orequal to a factor of about 5; about 10; about 30; about 100; about 300;about 1,000; about 3,000; about 10,000, about 100,000; or more. In someembodiments the discord in faradaic charge transfer can be in the rangeof from about 5 to about 100,000, or from about 5 to about 10,000, orfrom about 5 to about 1,000, or from about 5 to about 100, or from about10 to about 100,000, or from about 10 to about 10,000, or from about 10to about 1,000, or from about 10 to about 100, or from about 30 to about100,000, or from about 30 to about 10,000, or from about 30 to about1,000, or from about 30 to about 300, or from about 100 to about100,000, or from about 100 to about 10,000, or from about 100 to about1,000.

In electrochemical cells known in the art, there may be a difference inthe amount of byproduct formed at the counter electrode, relative to theamount of product formed at the working electrode when the half-cellreactions that occur at each of these electrodes requires a differingnumber of electrons. The difference between the amount ofelectrochemical product at the faradaic working electrode and theelectrochemical byproduct at the capacitive counter electrode isdetermined by the stoichiometry of the redox reactions involved.

Taking into consideration the discord in faradaic charge transfer, thepossibility that a different number of electrons can be required foreach of the half-cell reactions, and any other processes that affect theamount of byproduct that is formed, a discordantly smaller amount ofbyproduct is formed at the counter electrode if the ratio of product tobyproduct is greater than or equal to a factor of about 2; about 5;about 10; about 30; about 100; about 300; about 1,000; about 3,000;about 10,000, about 100,000; about 200,000 or more. For example, if theratio of the amount of faradaic charge transferred across the workingand counter electrodes is 30 and all the electrochemical reactions atthe counter electrode require 1 electron while all the electrochemicalreactions at the working electrode require 2 electrons, then the ratioof product to byproduct would be 15. In some embodiments, the ratio ofproduct to byproduct can be in the range of from about 2 to about200,000, or from about 2 to about 20,000, or from about 2 to about2,000, or from about 2 to about 200, or from about 5 to about 200,000,or from about 5 to about 20,000, or from about 5 to about 2,000, or fromabout 5 to about 200, or from about 15 to about 200,000, or from about15 to about 20,000, or from about 15 to about 2,000, or from about 15 toabout 600, or from about 50 to about 200,000, or from about 50 to about20,000, or from about 50 to about 2,000.

Coulometric methods can be used to add a small measurable amount of aspecies into a system, for example, into a nanosystem using anultramicroelectrode (UME). Because of the small size of nanosystems, theusual cell separators cannot easily be employed to prevent the undesiredproducts generated at the counter electrode from being introduced intothe system. Utilizing a system with a faradaic working electrode and acapacitive counter electrode can allow both electrodes to be in contactwith the electrolyte solution in a single cell, while reducing orpreventing the production of undesired products.

When the polarity of the electrical energy applied to the electrodes ischanged with time, current can flow alternately in both directions. Forexample, when the capacitive counter electrode charges to a largevoltage, the polarity of the electrical energy can be reversed todischarge the capacitive counter electrode. Optionally, the counterelectrode can be charged with the opposite polarity. During thisswitching process, the faradaic working electrode can act in turn as ananode and as a cathode (depending on the direction of current flow).While the use of two faradaic electrodes can cause both anodic andcathodic reactions to occur at the same time at differing places, thisinvention enables the anodic and cathodic reactions to occur atdiffering times at the same place. This temporal separation can beuseful in several circumstances.

B. Temporal Separation Methods

Temporal separation can be useful where the counter and workingelectrodes are sufficiently close that reaction products from oneelectrode could, if generated at the same time, diffuse to and interferewith the reaction at the other electrode. For example, in-situgeneration of chlorine gas from chloride ions in aqueous solution can becompromised by generation of hydroxy ions at a faradaic counterelectrode by the formation of hypochlorite. Through time separation, thedesired product (e.g., chlorine gas) can be used or removed (e.g., viadiffusion or convection) from the electrodes before the compromisingreaction occurs. The removal can be considered as a temporal-spatialseparation.

In some embodiments, the temporal-spatial separation can be created byflowing liquids past the electrodes. The liquid downstream of theelectrode can then comprise alternating cathodic and anodic reactionproducts. For example, a solution comprising dissolved oxygen gas andchloride ions can generate alternately chlorine gas and hydrogenperoxide downstream of the electrodes. This combination can be used, forexample, for decontaminating a device or cartridge. In some embodiments,temporal-spatial separations can be created by reaction productsdiffering in density (e.g., a gas and an ion). By giving the gas producttime to diffuse away from the working electrode before the ion iscreated, the reaction products can be separated.

In some embodiments, the temporally separated reaction products are notspatially separated; rather they can be alternately used in situ. Forexample, the pH of a reaction cell can be alternately adjusted up anddown by 1, 2, 3, 4, or more pH units by the faradaic creation of hydroxyor hydronium ions. Cycling the chemical environment in a region can haveseveral uses. For example, a new method of DNA amplification can be madecreating strand separation and renaturation cycles not by cyclingtemperature as is done in PCR, but by cycling the chemical environment.

C. Additional Embodiments of Apparatus and Assays Useful for CertainMethods

In some embodiments, the present invention is directed to an apparatuscomprising an electrochemical cell comprising: a faradaic workingelectrode; a capacitive counter electrode; and a container capable ofreceiving an electrolyte solution.

In some embodiments, the electrochemical cell at least partiallyencloses a sample volume.

In some embodiments, the electrochemical cell further comprises areference electrode.

In some embodiments, the apparatus further comprises anassay-performance-substance comprising a labeled binding partner for ananalyte or a labeled analog of the analyte.

In some embodiments, the apparatus further comprises a filter in fluidicconnection to the faradaic working electrode and the capacitive counterelectrode.

In some embodiments, the apparatus further comprises a source ofelectrical energy capable of being electrically connectable to thefaradaic working electrode and the capacitive counter electrode.

In some embodiments, the apparatus further comprises a photodetectorpositioned to detect light emitted on, at or near the faradaic workingelectrode.

In some embodiments, the apparatus further comprises a pump arranged tobe able to move liquid across or onto the faradaic working electrode.

In some embodiments, the present invention is directed to a method offor determining the presence or amount of an analyte in a samplecomprising the steps of:

(a) optionally preprocessing the sample;

(b) contacting a faradaic working electrode to a solution comprising theoptionally pre-processed sample; and an electrolyte;

(c) contacting a capacitive counter electrode to the solution;

(d) supplying electrical energy between the faradaic working electrodeand the capacitive counter electrode sufficient to provide for faradaiccharge transfer at the faradaic working electrode;

(e) measuring at least one of (i) light, (ii) current, (iii) voltage,and (iv) charge to determine the presence or amount of the analyte inthe sample.

In some embodiments, the sample is not pre-processed.

In some embodiments, the sample is pre-processed by filtering the samplewith a filter, wherein the filter has a pore size rating less than orequal to about 100 microns and greater than or equal to about 10 microns

In some embodiments, the sample is pre-processed by filtering the samplewith a filter, wherein the filter has a pore size rating less than orequal to about 10 microns and greater than or equal to about 1 microns.

In some embodiments, the sample is pre-processed by filtering the samplewith a filter, wherein the filter has a pore size rating less than orequal to about 1 micron and greater than or equal to about 0.1 microns.

In some embodiments, the sample is preprocessed by filtering the samplewith a filter, wherein the filter has a pore size rating less than orequal to about 0.1 micron and greater than or equal to about 0.02microns.

In some embodiments, the solution further comprises anassay-performance-substance comprising a labeled binding partner for ananalyte or a labeled analog of the analyte.

In some embodiments, the label of the labeled binding partner for theanalyte and/or the labeled analog of the analyte is the ECL moiety.

In some embodiments, the amount of analyte in the sample is determinedby activating the ECL moiety in solution by applying anelectrochemiluminescence-inducing electrical waveform to the faradaicworking electrode; and measuring the luminescence emitted by the ECLmoiety.

In some embodiments, the solution further comprises a second bindingpartner for the analyte wherein the second binding partner is linked toa support. In some embodiments, the support is a magnetizable bead.

In some embodiments, the method is a method for analyzing the sample formultiple analytes.

In some embodiments, the reagent that aids in the detection of theanalyte or the first binding partner is an electrolyte.

In some embodiments, the first binding partner is a labeled bindingpartner.

In some embodiments, the labeled binding partner comprises an ECLmoiety.

In some embodiments, the reagent that aids in the detection of theanalyte or the first binding partner is an ECL coreactant.

In some embodiments, the first binding partner is measured by measuringthe electrochemiluminescence emitted by the ECL moiety by:

(a) inducing the ECL moiety in solution to emit light by applying anelectrochemiluminescence-inducing electrical waveform to the workingelectrode in the presence of the ECL moiety; and

(b) measuring the luminescence emitted by the ECL moiety.

In some embodiments, the method further comprises the step of adding tofiltrate of step (b) a second binding partner for the analyte whereinthe second binding partner is linked to a support. In some embodiments,the support is a magnetizable bead.

In some embodiments, the method further comprises the step ofmagnetically collecting the magnetizable beads along the faradaicworking electrode surface through the use of a magnet.

In some embodiments, the faradaic current flow at the faradaic workingelectrode alternates in direction to reduce the potential across thecapacitive counter electrode.

In some embodiments, the present invention is directed to a method ofgenerating at least one electrochemical product at a working electrodewhile generating a discordantly smaller amount of electrochemicalbyproduct at a counter electrode, comprising:

contacting a faradaic working electrode with an electrolyte solution;

contacting a capacitive counter electrode with the electrolyte solution;and

applying electrical energy between the faradaic working electrode andthe capacitive counter electrode

wherein the faradaic charge transferred across the faradaic workingelectrode is at least about 10 times the faradaic charge transferredacross the capacitive counter electrode.

In some embodiments, the faradaic charge transferred across the faradaicworking electrode is at least about 100 times the faradaic chargetransferred across the capacitive counter electrode.

In some embodiments, the faradaic charge transferred across the faradaicworking electrode is at least about 1,000 times the faradaic chargetransferred across the capacitive counter electrode.

In some embodiments, the applied electrical energy alternates in thepolarity to form at least one oxidative product and at least onereductive product at the working electrode.

In some embodiments, the faradaic working and capacitive counterelectrodes are located in a flow cell and movement of the oxidative andreductive products away from the faradaic working electrode surface isfacilitated by a flow of the electrolyte solution through the flow cell.

In some embodiments, the rate of alternating the polarity of theelectrical energy applied to the electrodes is sufficient to alloweither the oxidative product to move away from the electrode before thereductive product is formed or to allow the reductive product to moveaway from the electrode before the oxidative product is formed.

In some embodiments, the electrolyte solution comprises dissolved oxygengas and chloride ions.

In some embodiments, the product of one of the oxidative half-cellreactions or the reductive half-cell reactions is a gas and the otherproduct is an ion.

In some embodiments, the oxidative product comprises chlorine gas andthe reductive product comprises hydrogen peroxide.

In some embodiments, the oxidative product and the reductive product areused as part of a decontamination process.

In some embodiments, the oxidative product and the reductive product isa gas and the other is an ion.

In some embodiments, the amount of the electrochemical product isincreased by exposing an area of the counter electrode to theelectrolyte solution that was previously unexposed to the electrolytesolution.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe above-described invention. It is understood that these examples inno way serve to limit the true scope of this invention, but rather arepresented for illustrative purposes. The following abbreviations havethe following meanings. If an abbreviation is not defined within theapplication, the abbreviation has its generally accepted meaning

μm=micrometers or micronsA=amperebpy=bipyridylcm=centimetere.g.=for exampleF=faradM=molarmin=minutemm=millimetermM=millimolarmV=millivoltsnA=nanoamperenF=nanofaradnm=nanometerpA=picoamperePMT=photomultiplier tubes=secondTPA=tri-n-propylamineUME=ultramicroelectrodeV=volts

Example 1 Measurement of Faradaic Current at a Working Electrode in anElectrochemical Cell Having a Capacitive Counter Electrode

A 25 μm diameter Pt wire (101) sealed in a glass UME (102) tip served asthe faradaic electrode and contacted a few mm diameter drop of deionized(MilliQ) water placed on the SiO₂ film (500 nm thick) on a singlecrystal Si wafer (100) with a thin insulating film of SiO₂ functioningas the capacitive electrode. Samples of SiO₂/Si were prepared atSEMATECH (Austin, Tex.) by chemical vapor deposition without furthertreatment. In experiments where a water drop was moved along the SiO₂surface under a bias as shown schematically in FIG. 1, a 2 mm diameterglass tube (103) was attached to the 25 μm Pt tip with parafilm to avoidwater leakage. The tube was slightly over filled so that a water drop(104) was formed at its top to contact the SiO₂ surface and the dropcould be moved horizontally to continuously make contact with a freshpart of the oxide surface. The total faradaic charge injected at the Ptelectrode is given by Q_(f)=∫i dt−Q_(c,Pt)=∫idt−C_(dl,Pt)A_(Pt)ΔE=C_(dl,Si)A_(Si)ΔE−C_(dl,Pt)A_(Pt)ΔE (1) where Q_(f)is the total faradaic charge injected, i, the total current, Q_(c,Pt),the capacitive charge at the Pt electrode, C_(dl,Pt), the integralcapacitance of the Pt electrode, C_(dl,Si) the integral capacitance ofthe Si electrode, A_(Pt) and A_(Si), the areas of Pt and Si electrodes,respectively, and ΔE, the applied bias.

Once the current became negligibly small, the external circuit wasdisconnected for about 8 s. When it was reconnected at the same bias,the charging current was significantly smaller as shown in FIG. 2,(inset) in which the initial sharp spike was an electronic artifactresulting from capacitive coupling. When the circuit was opened for alonger time, such as 15 to 20 s, the charging current after reconnectiondid not appear very different than that in the inset in FIG. 2. Thisdemonstrates that the electronic and ionic charges brought to the SiO₂interface by the external potential changed only slightly at opencircuit.

Example 2 Measurement of Faradaic Current at a Moving Working Electrodein an Electrochemical Cell Having a Capacitive Counter Electrode

Using the set up described in Example 1, the water drop (104) andassociated Pt UME (101) were moved across the SiO₂ surface under aconstant bias. Before movement, a bias of −1 V was applied to the tipand the surface became fully charged. Then, the tip was moved laterallyby pushing the translation stage (105) manually at a rate of roughly 1cm/s over a distance of about 0.5 to 4 cm without disconnecting the biasand the current increased as shown in FIG. 3. The current reached asteady state at the nA level, which was maintained by continuousexposure to fresh surface. The current did not drop until the movementended and the charging at this location approached saturation (FIG. 3).The contact area of the SiO₂ electrode and the water drop remainedessentially constant during the lateral tip movement. The steady statecurrent depended on how fast the fresh surface was contacted. At a tipmovement rate of 25 μm/s, controlled with an inchworm motor, the steadystate current was about 3 pA (the leakage current was not detectable(noise floor <1 pA) with such a thick oxide layer). The 25 μm movementof the water drop in 1 s generated a new contact area corresponding to acapacitance of ˜5 pF assuming a parallel capacitor with SiO₂ as thedielectric material. In other words, under a bias of 1 V neglectingpotential drops at the Pt electrode and the solution resistance, amaximum current of ˜5 pA should be obtained compared to the actualcurrent of 3 pA actually observed. At such a low rate of tipdisplacement, it took several minutes for the water drop to completelymove away from its previous spot. Probably the stored charge on bothinterfaces of SiO₂ (ions in solution and electronic charge in the Si)either did not move or moved too slowly to follow the water drop. Theobserved behavior did not depend on the polarity of the bias applied tothe system. When a supporting electrolyte such as 0.1M Na₂SO₄, wasintroduced, the system charged more quickly because of the decrease ofsolution resistance. However the basic features were the same.

The results shown in FIG. 3 could be reproduced many times at differentlocations. Similar results were also obtained as the tip traveled in astepwise, repeated stop-and-go mode as shown in FIG. 4. Each time, thecharging current increased as the water drop started to move, and asteady state charging current was seen and decreased when the water dropstopped moving.

The capacitive electrode's surface of SiO₂ was hydrophobic (contactangle with pure water about 87° C.) and no visible trace of water wasleft after the drop moved away from a spot. In some cases, the waterdrop was moved in a pre-designed pattern so that the location of eachstop could be revisited later. When the water drop was moved back tothose spots, which had been previously fully charged, after a period of10 to 20 min, no appreciable charge could be observed under the samecharging conditions (the charging current was comparable to the oneshown in the inset of FIG. 1), confirming that the charges remained attheir original spot and did not move anywhere else. Moreover, storedcharges were not discharged at a neighboring spot that was about 7.5 mmaway, indicating that there was no communication among stored charges atdifferent locations. Independent of the charging history, all of thespots could be restored to their initial state by discharging the Siunder short circuit conditions. These findings agree with earlierstudies of the emersion of a metal electrode from solution into highvacuum, which indicated that the solution-formed double layer stillexisted in the high vacuum chamber after charging of the metal electrodein solution and then transfer to vacuum under a bias. See Hansen, etal., J. Electroanal. Chem., 1978, 93, 87 for further details.

A typical steady charging current was seen when the tip was stationaryand the potential was scanned at a rate of 100 mV/s as shown in theinset of FIG. 3. The calculated capacitance (C=i/v, where i and v arecurrent and scan rate, respectively) of 0.577 nF corresponding to 6.9nF/cm² from this result fitted a value for an ideal parallel capacitorusing SiO₂ as the dielectric material (C=_(∈∈) _(o) A/d, where ∈ is therelative permittivity of the dielectric material, 3.9 for SiO₂; ∈_(o)the permittivity of space; A the area, and d is 500 nm, the thickness ofSiO₂) with a diameter of 3.2 mm, which closely matched the actual areacontacted by the drop.

Example 3 Measurement of ECL at in an Electrochemical Cell Having aCapacitive Counter Electrode

A 250 μm Pt wire was bent at a right angle, coated with epoxy cement andthen polished to expose an area of about 0.02 mm² facing aphotomultiplier tube (PMT, R4220p, Hamamatsu, Bridgewater, N.J.). Apiece of Si with an area of ˜40 cm² and coated with a ˜50 nm thick SiO₂film was used the counter electrode in an aqueous solution containing0.5 mM Ru(bpy)₃ ²⁺[tris(2,2′-bipyridine)ruthenium(II)] in 0.10 Mtri-n-propylamine (TPA) with 0.10 M Tris/0.10 M LiClO₄ buffer (pH=8). AnAutolab potentiostat (Model PGSTAT100, EcoChemie, Utrecht, TheNetherlands) was used to control the applied potential with Pt as theworking electrode and the Si back contact as the counter electrode. Thereference electrode input on the potentiostat was connected to thecounter electrode. The ECL emission and the current were recordedsimultaneously during the measurement. Potential pulses from 1.4 V (30s) to −0.5 V (20 s) were applied to the system Pt/solution/SiO₂/Si. In aseparate measurement to obtain an ECL image, a 25 μm Pt UME tip was usedwith the same solution over a 9 cm² Si/SiO₂ surface mounted on the stageof an inverted microscope (Nikon, Model TE300, Melville, N.Y.). Foradditional experimental options see also Bard, A. J.; Ed.,Electrogenerated Chemiluminescence, Marcel Dekker, New York, 2004 andMiao, et al., J. Am. Chem. Soc., 2002, 124, 14478 and referencestherein.

ECL was generated by potential steps applied between a 0.02 mm·sup·2 Ptelectrode and a Si/SiO₂ (40 cm²) electrode. As shown in FIG. 5, ECL wasdetected immediately following the application of a potential pulse of1.4 V to the Pt electrode, triggering a negative charging process at theSiO₂ interface. As the charging current decreased, the ECL emissionintensity decreased. The decreasing current in this case representscharging of the counter electrode rather than the usual reactantdepletion at the Pt electrode. As expected, no ECL was seen under anegative bias of −0.5 V, which served, however, to discharge theinterface at the Si/SiO₂ electrode (with a corresponding faradaicreaction at the Pt electrode). This cycle could be repeated many timesand the ECL intensity decreased slightly each time probably due to thedepletion of active species near the Pt electrode surface. ECL was notdetected at the blocked electrode whose leakage current was thereforenegligible. In a different measurement with a 25 μm Pt tip over a 9 cm²Si/SiO₂ electrode with the same Ru(bpy)₃ ²⁺/TPA solution under aconstant bias of 1.4 V, an ECL image of the Pt tip was clearly seen withan inverted microscope as shown in the inset in FIG. 5. The productionof ECL provides clear evidence for a faradaic process in the singleelectrode electrochemical system and shows that ECL can be generated ina microcell without interference from counter electrode reactions.

All references cited herein are incorporated by reference in theirentirety. To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The foregoing description of possible implementations consistent withthe present invention does not represent a comprehensive list of allsuch implementations or all variations of the implementations described.The description of only some implementation should not be construed asan intention to exclude other implementations. Artisans will understandhow to implement the invention in the appended claims in many otherways, using equivalents and alternatives that do not depart from thescope of the following claims. Moreover, unless indicated to thecontrary in the preceding description, none of the components describedin the implementations is essential to the invention.

What is claimed is:
 1. A method of generating at least oneelectrochemical product at a working electrode while generating adiscordantly smaller amount of electrochemical byproduct at a counterelectrode, comprising the steps of: contacting a faradaic workingelectrode with an electrolyte solution; contacting a capacitive counterelectrode with the electrolyte solution; and applying electrical energybetween the faradaic working electrode and the capacitive counterelectrode; wherein the faradaic charge transferred across the faradaicworking electrode is at least about 10 times the faradaic chargetransferred across the capacitive counter electrode, thus generating adiscordantly smaller amount of electrochemical byproduct at the counterelectrode.
 2. The method of claim 1, wherein the faradaic chargetransferred across the faradaic working electrode is at least about 100times the faradaic charge transferred across the capacitive counterelectrode.
 3. The method of claim 1, wherein the faradaic chargetransferred across the faradaic working electrode is at least about1,000 times the faradaic charge transferred across the capacitivecounter electrode.
 4. The method of claim 1, wherein the appliedelectrical energy alternates in the polarity to form at least oneoxidative product and at least one reductive product at the workingelectrode.
 5. The method of claim 1, wherein the faradaic working andcapacitive counter electrodes are located in a flow cell and movement ofthe oxidative and reductive products away from the faradaic workingelectrode surface is facilitated by a flow of the electrolyte solutionthrough the flow cell.
 6. The method of claim 1, wherein the rate ofalternating the polarity of the electrical energy applied to theelectrodes is sufficient to allow either the oxidative product to moveaway from the electrode before the reductive product is formed or toallow the reductive product to move away from the electrode before theoxidative product is formed.
 7. The method of claim 1, wherein theelectrolyte solution comprises dissolved oxygen gas and chloride ions.8. The method of claim 7, wherein the oxidative product compriseschlorine gas and the reductive product comprises hydrogen peroxide. 9.The method of claim 1, wherein the oxidative product and the reductiveproduct are used as part of a decontamination process.
 10. The method ofclaim 1, wherein one of the oxidative product and the reductive productis a gas and the other is an ion.
 11. The method of claim 1, wherein theamount of the electrochemical product is increased by exposing an areaof the counter electrode to the electrolyte solution that was previouslyunexposed to the electrolyte solution.